MST1 modulation of apoptosis in cardiac tissue and modulators of MST1 for treatment and prevention of cardiac disease

ABSTRACT

The present invention relates to methods and agents for treatment, amelioration and prevention of cardiac disease, including cardiac myopathy, chronic heart failure and for management and reduction of cardiac myocyte death which may occur in response to ischemia/reperfusion or following myocardial infarction or other injury to the heart. The invention relates to methods for screening cardiotherapeutic compounds, including compounds which modulate cardiac myocyte apoptosis, particularly targeting Mst1 and the Mst1 pathway. The present invention further encompasses compounds identified by such screening methods and compositions comprising these compounds. The invention also provides methods for treatment, amelioration and prevention of cardiac disease comprising administering compounds or agents which modulate, particularly inhibit, Mst1 or the Mst1 kinase pathway, including administering a nucleic acid encoding an altered form of Mst1, particularly a dominant negative Mst1, which acts as an antagonist of Mst1.

RELATED APPLICATIONS

The present application claims the benefit of the filing date pursuantto 35 U.S.C. § 119 of provisional application Ser. No. 60/418,002, filedOct. 11, 2003, which is incorporated herein by reference in itsentirety.

GOVERNMENTAL SUPPORT

The research leading to the present invention was supported, at least inpart, by a grant from the National Institutes of Health, Grant NumbersHL-59139,HL-33107, HL-33065, HL65182, HL-65183, AG-14121, HL-69020,HL-67724 and HL-67727. Accordingly, the Government may have certainrights in the invention.

FIELD OF THE INVENTION

The present invention relates to methods and agents for treatment,amelioration and prevention of cardiac disease, including cardiacmyopathy, chronic heart failure and for management and reduction ofcardiac myocyte death which may occur in response toischemia/reperfusion or following myocardial infarction or other injuryto the heart. The invention relates to methods for screeningcardiotherapeutic compounds, including compounds which modulate cardiacmyocyte apoptosis, particularly targeting Mst1 and the Mst1 pathway. Thepresent invention further encompasses compounds identified by suchscreening methods and compositions comprising these compounds.

BACKGROUND OF THE INVENTION

Intensive studies in the past decade have shown that cardiac myocytedeath, which has characteristics of apoptosis, occurs in response toischemia/reperfusion (I/R) and during cardiac remodeling aftermyocardial infarction (1-9). Although controversies still existregarding the occurrence of apoptosis-vs oncosis in the ischemic heart(10,11), it seems established that the signaling mechanisms promotingmyocyte cell death, including caspases, are activated by I/R and incardiomyopathy (12-16) (reviewed in 47). Recent evidence suggests thatmyocyte proliferation could take place in response to the loss ofcardiac myocyte after myocardial infarction (17). However, consideringthe limited capacity of terminally differentiated cardiac myocytes forproliferation, it is important to understand how these cell deathpromoting signaling mechanisms are activated by I/R and in myopathichearts in order to establish interventions efficiently preventing thecell loss in various stages of heart diseases (10). It has been shownthat intracellular stress-responsive protein kinases, including JNKs andp38-MAPKs, are activated by I/R in the heart (18-23). Although thesekinases are likely to affect both cell death and cell survival, theroles of the stress-responsive protein kinases (SRPKs) in cardiacmyocyte apoptosis are not fully understood. Furthermore, the SRPKselectively promoting cardiac myocyte apoptosis has not been identifiedin vivo.

Mst1 (mammalian sterile 20-like kinase-1) is a ubiquitously expressedserine/threonine kinase (24,25), which belongs to a mammalian sterile 20(STE 20)-like kinase family consisting of Pak1, Mst1, Mst2, Khs, Gck,Sok1, Nik, Hpk1 and Sps1 (26,27). Increasing lines of evidence suggestthat Mst1 and other STE20-like family kinases play an important role inmediating apoptosis (reviewed in (27)). Mst 1 is activated by somepro-apoptotic stimuli in fibroblastic and lymphocytic cell lines.However, stimuli shown to activate Mst1 are generally limited tonon-physiological stresses, including genotoxic compounds and extremeheat shock (25,28-31). Mst1 can be an efficient mediator of apoptosisbecause it is cleaved by caspases and this cleavage increases kinaseactivities of Mst1, which in turn activates caspase 3 (32), therebyconstituting a powerful amplification loop of apoptotic response (33).Importantly, however, whether or not activation of Mst1 is required forin vivo cell death in response to clinically relevant pathologic insultshas not been determined in any organs, including the heart.

Despite a growing understanding that apoptosis plays a physiologicalrole and potentially pathological role in the heart there still exists asignificant need in the art for methods and agents for treatment,amelioration and prevention of cardiac disease, including cardiacmyopathy, chronic heart failure and for management and reduction ofcardiac myocyte death which may occur in response toischemia/reperfusion or following myocardial infarction or other injuryto the heart.

The citation of references herein shall not be construed as an admissionthat such is prior art to the present invention.

SUMMARY OF THE INVENTION

In accordance with the present invention, it has been recognized anddemonstrated that stimulation or enhanced expression of Mst1 causescardiac myocyte apoptosis and dilated cardiomyopathy, without anycompensatory cardiac myocyte hypertrophy. In addition, specificinhibition of endogenous Mst1, for instance by dominant negative Mst1,inhibits cardiac myocyte apoptosis and myocardial infarction in responseto ischemia/reperfusion (I/R). Inhibition of endogenous Mst1 alsoinhibits the negative cardiac effects of cardiotoxic drugs, includingdoxorubicin. In addition, on inhibition of endogenous Mst1, the cardiacdilation and cardiac dysfunction following myocardial infarction aresignificantly reduced. Thus, Mst1 plays an essential role in mediatingapoptosis by clinically relevant pathologic stimuli in the heart andrepresents an important therapeutic target in ischemic heart diseases.

The invention relates to the application and use of modulators,including inhibitors, of Mst1 to treat or prevent heart disease. Theinvention relates to the use of modulators, particularly inhibitors, ofthe Mst1 kinase for amelioration, treatment or prevention of cardiacdisease, particularly wherein it is desired to reduce or control cardiacmyocyte apoptosis, including in congestive heart failure,cardiomyopathy, including ischemic and nonischemic cardiomyopathy,coronary artery disease, arrhythmias, fibrosis of the heart, valvedefects, and atherosclerosis, as well as in instances where facilitationof enhanced heart function or maintenance of cardiac myocytes isdesired.

In a particular aspect, the invention provides methods of modulating theapoptosis of cardiac myocytes and cardiac myopathy in a mammal byadministration of a compound or agent that blocks or otherwise inhibitsMst1 or the Mst1 pathway. In one aspect, a method for modulating theapoptosis of cardiac myocytes and cardiac myopathy is provided wherebyan effective amount of an inhibitor of Mst1 is administered.

In a further aspect, the invention provides a method for treatingcardiac disease in a mammal comprising administering to said mammal aneffective amount of an Mst1 inhibitor. In a particular aspect, theinvention further provides a method for treating a cardiac disease ordysfunction selected from the group of congestive heart failure,cardiomyopathy, including ischemic and nonischemic cardiomyopathy,coronary artery disease, arrhythmias, fibrosis of the heart, valvedefects, and atherosclerosis in a mammal comprising administering tosaid mammal an effective amount of an Mst1 inhibitor. In a still furtheraspect a method is provided for reducing the risk of cardiomyopathy ordysfunction in a mammal after said mammal has suffered a myocardialinfarct or other coronary event wherein blood flow to the heart iscompromised or significantly reduced comprising administering to saidmammal an effective amount of an Mst1 inhibitor or Mst1 pathwayinhibitor.

In a further aspect, the invention provides a method ofcardioprotection, wherein an inhibitor of Mst1 is administered inconjunction with or following therapy with a compound or drug which iscardiotoxic or has potential to be cardiotoxic. In this method, an Mst1modulator blocks or otherwise reduces the cardiotoxic effects of a drugor compound. In a particular embodiment, the drug or compound is achemotherapeutic agent, particularly an anti-cancer agent, including butnot limited to doxorubicin. In a further aspect, administration of theMst1 modulator enables chemotherapeutic, particularly anti-cancer oranti-tumor compounds, treatment without significant cardiotoxic effectsor permits prolonged chemotherapy or administration of higherconcentrations or amounts of a potentially cardiotoxic drug.

In a further aspect, the invention provides a method for treatingcardiac disease in a mammal comprising administering to said mammal aneffective amount of an Mst1 inhibitor in combination with one or moreother compounds for the treatment of heart disease, including but notlimited to congestive heart failure, cardiomyopathy, including ischemicand nonischemic cardiomyopathy, coronary artery disease, arrhythmias,fibrosis of the heart, valve defects, and atherosclerosis. Inparticular, an effective amount of an Mst1 inhibitor may be used incombination with one or more compound for treatment of cardiac diseaseor of atherosclerosis, including but not limited to a beta-blocker,nitrate, calcium channel antagonists, angiotensin-converting enzyme(ACE) inhibitors, an anti-platelet drug, diuretics, digoxin andantilipemic agents, agents which alter cholesterol or lipid metabolism.Further examples of such or treatmantilipemic agents include, but arenot limited to the statins, such as Lovastatin (Mevacor®), Atorvastatin(Lipitor®), Simvastatin (Zocor®), Cerivastatin (Baycol®), Pravastatin(Pravacol®), Fluvastatin (Lescol®) and Rosuvastatin; as well as otherclasses of compounds including cholesterol ester transfer proteininhibitors; cholesterol adsorption inhibitors (e.g., Ezetimide®);fibrates (e.g., Gemfibrozil®); nicotinic acid (e.g., niacin and analogs,Niaspan®); bile acid reuptake inhibitors; and modulators of nuclearhormone receptors such as PPARa, PPARb, PPARg, LXRa, LXRb, FXR, RORa andSHP.

The object of the invention extends to the provision of methods forscreening for cardiotherapeutic compounds, particularly compounds whichmodulate cardiac myocytes, particularly apoptosis of cardiac myocytes,by using Mst1 or a member of the Mst1 pathway. In a particular object,methods are provided for screening compounds which modulate cardiacmyocytes, by modulating Mst1, Mst1 kinase activity, or the Mst1 pathway,for instance by modulating caspase which activates Mst1 bycaspase-mediated cleavage of the C-terminal inhibitory domain. Inaddition, the present invention encompasses compounds that areidentified by the screening methods disclosed herein. Further providedare methods and compositions for modulating cardiac myocytes and therebymodulating cardiac disease and cardiac function.

A method of the invention involves screening cardiotherapeutic compoundsand includes the steps of selecting compounds that modulate Mst1 or someportion of the Mst1 pathway, and performing assays with said compounds.The method of screening of the present invention may also include thestep of identifying compounds that lead to reduction of cardiac myocyteapoptosis, or alternatively, to a decrease in levels or activity ofMst1. Selecting compounds that modulate Mst1 or the Mst1 pathway mayinvolve different assays, such as, e.g., phosphorylation assays whereina compound's ability to block phosphorylation of or by Mst1 or enhancedephosphorylation of Mst1 or a Mst1 target is determined directly orwherein the activity of a molecule downstream of Mst1 is determined. Forinstance, the phosphorylation of the Mst1 target myelin basic protein(MBP) can be assayed, including by incubation with labeled phosphate(e.g. γP³²) and determining whether MBP is labeled. Compounds forscreening may be selected from various libraries of small molecularweight compounds, peptides, or alternatively may be selected by homologymodeling, computational modeling, and screening phage display libraries.

The invention thus includes an assay system for screening of potentialdrugs effective to modulate Mst1 activity of cardiac cells byinterrupting or potentiating Mst1 or the Mst1 pathway. In one instance,the test drug could be administered to a cellular sample to determineits effect upon the kinase activity or phosphorylation status of Mst1 oran Mst1 target, by comparison with a control.

The present invention also includes compositions for treating orameliorating cardiac disease, particularly for modulating apoptosis ofcardiac myocytes. These compositions may comprise compounds identifiedby the screening methods disclosed herein. Thus, these compositions maybe used to cardiac diseases or conditions where cardiac function orefficiency is compromised.

It is a thus an object of the present invention to providepharmaceutical compositions for use in therapeutic methods formodulation of cardiac disease which comprise an Mst1 inhibitor. In afurther aspect, the invention provides pharmaceutical compositions foruse in therapeutic methods for modulation of cardiac disease whichcomprise an Mst1 inhibitor and one or more compounds for the treatmentof heart disease, including but not limited to congestive heart failure,cardiomyopathy, including ischemic and nonischemic cardiomyopathy,coronary artery disease, arrhythmias, fibrosis of the heart, valvedefects, and atherosclerosis. In particular, an effective amount of anMst1 inhibitor may be used in combination with one or more compound fortreatment of cardiac disease or of atherosclerosis, including but notlimited to a beta-blocker, nitrate, calcium channel antagonists,angiotensin-converting enzyme (ACE) inhibitors, an anti-platelet drug,diuretics, digoxin and antilipemic agents, agents which altercholesterol or lipid metabolism.

In a particular aspect of the invention, the Mst1 inhibitor is a mutantMst1 which acts as an antagonist of Mst1, particularly a dominantnegative Mst1. In one such example, the Mst1 mutant is Mst1, (K59R). Ina method of the invention, cardiac myocytes are transduced, transfectedor infected with nucleic acid or vector containing nucleic acid encodinga mutant Mst1 which acts as a dominant negative to antagonize wild typeand endogenous Mst1. By expression of dominant negative Mst1 in cardiacmyocytes, apoptosis is blocked or reduced and cardiac disease is therebytreated or ameliorated.

The DNA sequences of Mst1 of use in the present invention or portionsthereof, may be prepared with a variety of known vectors, particularlyvectors capable of expressing such DNAs in animal cells, particularly inhuman cells. In one such embodiment, the vectors include those suitablefor delivery to human cells, particularly to cardiac or heart cells,such as myocytes. Examples of such vectors include viral vectors such asadenovirus, or naked DNA vectors, for instance for injection or deliveryto the heart wherein the DNA is then expressed. The present inventionalso includes the preparation of plasmids or viruses including suchvectors, and the use of the DNA sequences to construct vectorsexpressing antisense RNA, small interfering RNAs or ribozymes whichwould attack or interfere with the mRNAs of any or all of the DNAsequences encoding native or endogenous Mst1. Correspondingly, thepreparation of antisense RNA, siRNAs and ribozymes are included herein.

In a further object, the invention provides animal models, includingtransgenic models, of cardiac disease and cardiac myopathy. Such animalmodels include transgenic animals expressing enhanced amounts of Mst1 oraltered Mst1 (including wherein Mst1 is substantially more active orconstitutively active). These animals are useful in studies of cardiacdisease, including for the screening and assessment of potentialcardiotherapeutic compounds. These animals include but are not limitedto rats, mice, pigs, chicken, cows, monkeys, rabbits, sheep and dogs.

Other objects and advantages will become apparent to those skilled inthe art from a review of the following description which proceeds withreference to the following illustrative drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, 1C, 1D and 1E depict Mst1 activity assessed on treatmentof cardiac myocytes with chelerythrine (Chele) or calyculin A. (A)Cardiac myocytes were treated with indicated concentrations ofchelerythrine for 1 h. Cell lysates were subjected to in-gel myelinbasic protein (MBP) kinase assay as well as immunoblotting withanti-cleaved caspase-3 antibody. Results are representative of more thanfive experiments. (B) Cardiac myocytes were treated with chelerythrine(Chele). In the right panel, myocytes were transduced with eithercontrol virus or adenovirus harboring XIAP 48 hours before Cheleapplication. Cell lysates were subjected with immune complex in-gel MBPkinase assays, using anti-Mst1 polyclonal antibody (pAb-15). Theactivity of cleaved Mst1 is shown. (C) Cardiac myocytes were treatedwith Chele for the indicated durations. Immunoblot analyses wereconducted by using anti-Mst1 monoclonal antibody (upper panel) andanti-Mst1 polyclonal antibody (lower panel), which detects the fulllength form and cleaved form (amino-terminal half) of Mst1,respectively. (D) Cardiac myocytes were subjected to 8h of hypoxia alone(H) or 8h hypoxia plus 12 h of reoxygenation (H/R). In-gel MBP kinaseassays were performed. Control, C. Upper arrow indicates the full lengthform of Mst1, while lower arrow cleaved form of Mst1. (E) Cardiacmyocytes were treated with vehicle (lane 1) or calyculin A (1 μM, lane2). In-gel MBP kinase assays were performed. Arrow indicates the fulllength form of Mst1.

FIGS. 2A, 2B, 2C, 2D, 2E, 2F and 2G. (A to G) Cardiac myocytes weretransduced with adenovirus harboring either wild type Mst1 (AdX-Mst1),Mst1 (K59R) (AdX-Mst1(K59R)) or control adenovirus (Ad5 βgal) atindicated concentrations. Myocytes were harvested 48 h aftertransduction. Some myocytes (C and F) were treated with a caspase-3inhibitor (DEVD-CHO, 100 □M). (A and B) Immunoblot analyses wereperformed by using anti-Mst1 polyclonal antibody. MOI, multiplicity ofinfection. Cont, control where no virus was applied. (C) In-gel MBPassays were performed. (D) The effect of adenovirus transduction (30MOI) upon the morphology of cardiac myocytes is shown. Note that celldeath with shrinkage is observed in AdX-Mst1 transduced cardiacmyocytes. (E and F) Cytoplasmic accumulation of mono- andoligo-nucleosomes, a sensitive indicator of DNA fragmentation byapoptosis, was quantitated by Cell Death ELISA Plus. n=3 (F) (G)Activation caspase-3 was determined by immunoblot analyses withanti-cleaved caspase-3 antibody. n=3.

FIG. 3 depicts the effect of dominant negative Mst1 uponchelerythrine-induced cardiac myocyte apoptosis. Cardiac myocytes weretransduced with er control virus or adenovirus harboring dominantnegative Mst1 (Mst1(K59R)). Forty-eight hours after transduction,cardiac myocytes were treated with chelerythrine (10 μM) for 1 h andcytoplasmic accumulation of mono- and oligo-nucleosome was quantitated.n=3.

FIGS. 4A, 4B, 4C, 4D, 4E, 4F, 4G, 4H, and 4I. (A) Immunoblot analyses ofthe heart homogenates with anti-myc antibody. (B) Tissue homogenateswere prepared from various organs of Tg-Mst1. Immunoblot analyses wereperformed with anti-myc antibody. (C) Heart homogenates were preparedfrom Tg-Mst1 or non-transgenic control mice (Ntg). In-gel MBP kinaseassays were performed. (D) Gross appearance and a transverse section ofthe hearts obtained from Tg-Mst1 and NTg. Hematoxylin Eosin staining wasperformed (3 months old). (E) A photograph of the liver isolated fromTg-Mst1 and the littermate NTg (3 months old). (F) Picric acid siriusred staining of heart sections obtained from Tg-Mst1 and NTg (1.5 monthold). (G) LV cardiac myocyte cross sectional area was obtained fromTg-Mst1 and NTg as described in the Method section. Seven Tg-Mst1 and 4NTg mice were used for the analysis. (H) Heart homogenates were preparedfrom Tg-Mst1 and NTg. (I) Immunoblot analyses were performed by usinganti-cleaved caspase-3 antibody. n=3.

FIG. 5 depicts myocyte length and contractile (% contraction) andrelaxation (TR 70%: time for 70% relengthening) function. Myocyte lengthwas significantly reduced, p<0.05, in myocytes from Tg-Mst1 (n=4, 111cells) compared with WT (n=4, 129 cells). There was no change incontractile and relaxation function.

FIGS. 6A, 6B, 6C, 6D and 6E depicts (A) Immunoblot analyses of the hearthomogenates with anti-myc antibody. In (B)-(E), Tg-DN-Mst1 ornon-transgenic control mice (NTg) were subjected to 20 min ischemia and24 h reperfusion or sham operation. (B) The heart homogenates (100 μg)obtained from ischemic (I) and non-ischemic (N) areas of the leftventricle (LV) or from intact LV of the sham operated mice weresubjected to in gel myelin basic protein (MBP) kinase assays.Ischemia/reperfusion (I/R) increased kinase activities of Mst1 in theischemic area of NTg mice, while activation of Mst1 by I/R wascompletely abolished in Tg-DN-Mst1. (C) The effect of I/R upon theextent of LV myocardial infarction (MI) in Tg-DN-Mst1 and NTg controlmice. The MI area/area at risk (AAR) was determined as described in theMethod section. Note that MI area/AAR was significantly smaller inTg-DN-Mst1 compared with that in NTg. (D) LV tissue sections weresubjected to TUNEL staining and DAPI staining. n=11. (E) Genomic DNA wasisolated from non-ischemic (N) and ischemic (I) areas and DNA ladderingassays were performed. The extent of DNA laddering in response to I/Rwas significantly smaller in Tg-DN-Mst1 compared with that in NTg. n=3.

FIGS. 7A, 7B, 7C and 7D depicts (A) Cardiac myocytes were transducedwith either control virus (Cont) or AdX-Mst1(Mst1) at 10 MOI for 48 h.As positive control, myocytes were treated with chelerythrine (Chele, 10μM) for 1 h. The mitochondria-free cytosolic fraction was obtained.Western blot analysis was performed using anti-cytochrome c antibody(Pharmingen, San Diego, Calif.). Cytochrome c oxidase IVimmunoreactivity was negligible in these samples. Chele caused a releaseof cytochrome c to the cytosolic fraction. Expression of Mst1 increasedrelease of cytochrome c. n=3. (B,C) Cardiac myocytes were transducedwith either control virus or AdX-Mst1(Mst1) at indicated MOIs for 48 h.(upper) Immunoblot analyses were conducted using anti-phospho p38-MAPKantibody (B) or anti-phospho JNK antibody (C). (lower) The filters werere-probed with anti-p38-MAPK antibody (B) or anti-JNK1 antibody (C). Inb and c, Similar results were obtained in 4 experiments. (D) Cardiacmyocytes were transduced with either control virus or AdX-DN-Mst1 virus.Myocytes were then stimulated with or without chelerythrine (Chele, 10μM) for 60 min. Myocyte lysates were subjected to immunoblot analysisusing anti-phospho p38-MAPK antibody. The filter was re-probed withanti-p38-MAPK antibody. Similar results were obtained in 3 experiments.

FIGS. 8A and 8B depicts mRNA expression of PGC-1α and β is downregulatedin Tg-Mst1 (PGC-1 is a nuclear transcription co-factor, which plays animportant role in regulating expression of nuclear encoded mitochondrialgenes). RT-PCR results are shown. Each band in the gel shown in (A)represents RNA combined from 2 separate hearts. Mice were approximately2 months old. Data represents a single experiment. The relative RNAlevels are graphed in (B).

FIGS. 9A and 9B depicts that protein expression of cytochrome c oxidaseis downregulated in Tg-Mst1 animals. Western blots of protein fromnontransgenic (N) and transgenic (T) individual animals are shown in (A)for cytochrome c oxidase VIIa (COX VIIa), IV (COX IV) and Vb (COX Vb).Relative protein expression for each COX is graphed in (B). Data aremean ±SEM.

FIG. 10 depicts neonatal rat cardiac myocytes after 6 hour stimulationwith Doxorubicin (Doxo). Control myocytes show no evidence of shrinkage,vacuolization, or pyknosis. After 6 hours of Doxo induction, myocytesshow a dose dependent cell shrinkage and nuclear pyknosis.

FIG. 11 ELISA shows a reduction of mono- and oligo-nucleosome content inthe Adx-Dn-Mst1 treated group after 6 hrs of Doxo stimulation. OD 405, 5moi of control virus (Lac Z) and DN-Mst1 virus (DNV) respectively. Mostlikely the slight decrease in the DNV Doxo 1 mM lane is due to increasednecrosis of cells in the wells at this dose.

FIG. 12 depicts a cell death ELISA which again shows a trend in thereduction of mono- and oligo-nucleosome content in the Adx-Dn-Mst1treated group after 6 hrs of Doxo stimulation. OD 405 with 10 moi of LacZ and DN-Mst1 AdX respectively.

FIG. 13 provides a Western blot depicting cleavage of caspase-3 in thepresence of Doxo at the 6 and 24 hour time points. Data suggest thatDoxo treatment activates caspase-3 in a time dependent manner.

FIG. 14 shows a stripped membrane of the previous (FIG. 13) CC-3antibody reprobed with CC-9 antibody. This western blot also depictscleavage of caspase-9 in the presence of Doxo at the 6 and 24 hour timepoints, with the stronger band appearing at the 24 hour time point. Datasuggest that Doxo treatment activates caspase-9 in a time dependentmanner.

FIG. 15 provides an in gel kinase assay depicting activation of the fulllength (61kD) and cleaved (34 kD) forms of Mst1 after 6 hr Doxostimulation of cardiac myocytes. Myelin basic protein is a majorsubstrate that binds Mst1 during phosphorylation.

FIG. 16 depicts a Western blot with MST1 antibody after 6 hrs ofstimulation of neonatal cardiac myocyte culture with Doxo, showingcleaved Mst-1 at the 0.5 mM dose. Doxo activates cleaved Mst1 but in thepresence of DN-Mst1 this reaction is suppressed.

FIG. 17 depicts an immunoblot of cardiac myocytes exposed to 10 moi ofeither βgal (Lac Z) or DN-Mst1 adenovirus prior to all myocytes beingexposed to 6 hr of Doxo stimulation. In the presence of the DN virus,there is no activation of cleaved caspase −3 when compared to βgal anduninfected myocytes stimulated with Doxo 0.5 mM.

FIG. 18 compares 10 moi control virus (βgal) and 10 moi Adx-DN-Mst1 atbaseline and after 6 hrs of stimulation with Doxo. Cell death andshrinkage are noted in the control virus specimen treated with Doxo butnot in the Adx-DN-Mst1 treated specimen. This suggests that DN-Mst1inhibits cardiac myocyte apoptosis by Doxo.

FIG. 19 compares control and dominant negative transgenic mice with andwithout Doxo injection. Dominant negative transgenic mice receiving theDoxo injection maintain ejection fraction post Doxo injection whencompared to their non-transgenic litermates. This suggests that Dn-Mst1confers a protective effect, possibly inhibiting apoptosis socontractility is maintained.

FIG. 20 compares control and dominant negative transgenic mice with andwithout Doxo injection. Dominant negative transgenic mice receiving theDoxo injection maintain their % fractional shortening post Doxoinjection when compared to their non-transgenic litermates. Thissuggests that Dn-Mst1 confers a protective effect, possibly byinhibiting apoptosis to maintain contractility.

FIG. 21 compares the body weights of the non-transgenic an DN-Mst1 micepre and post injections of Doxo and normal saline. The significant lossof weight between the NTG pre and DN-TG post Doxo injection mice is mostlikely due to the well known side effect of anorexia that accompaniesDoxo treatment.

FIG. 22 compares the LVEDD of the non-transgenic and DN-Mst1 mice preand post Doxo/normal saline treatment. The p value is not significant.

FIGS. 23A and 23B depict trangsenic Mst1 and nontransgenic animals 4weeks after left coronary artery ligation to generate a myocardialinfarction (MI) in vivo. (A) indicates that a similar size of myocardialinfarction was created by ligation of the left coronary artery innon-transgenic (NTG) and transgenic (Tg-DN-Mst1) mice. LVEDD (B) andLVEF (B) were measured in sham operated and MI mice.

DETAILED DESCRIPTION

The studies provided herein assessed whether or not Mst1 plays anessential role in mediating apoptosis in cardiac myocytes, by using bothcultured cardiac myocytes in vitro and transgenic mouse models in vivo.The results demonstrate that Mst1 is activated not only by genotoxiccompounds but also by clinically relevant pathologic insults in theheart. Stimulation of or enhanced expression of Mst1 causes cardiacmyocyte apoptosis and dilated cardiomyopathy, without any compensatorycardiac myocyte hypertrophy. In addition, specific inhibition ofendogenous Mst1 by dominant negative Mst1 inhibits cardiac myocyteapoptosis and myocardial infarction in response to ischemia/reperfusion(I/R). Inhibition of endogenous Mst1 also inhibits the negative cardiaceffects of cardiotoxic drugs, as demonstrated in blockage ofdoxorubicin-induced cardiomyopathy. In addition, on inhibition ofendogenous Mst1, the cardiac dilation and cardiac dysfunction followingmyocardial infarction are significantly reduced. Thus, Mst1 plays anessential role in mediating apoptosis by clinically relevant pathologicstimuli in the heart and represents an important therapeutic target inischemic heart diseases.

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook et al, “Molecular Cloning:A Laboratory Manual” (1989); “Current Protocols in Molecular Biology”Volumes I-III [Ausubel, R. M., ed. (1994)]; “Cell Biology: A LaboratoryHandbook” Volumes I-III [J. E. Celis, ed. (1994))]; “Current Protocolsin Immunology” Volumes I-III [Coligan, J. E., ed. (1994)];“Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic AcidHybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “TranscriptionAnd Translation” [B. D. Hames & S. J. Higgins, eds. (1984)]; “AnimalCell Culture” [R. I. Freshney, ed. (1986)]; “Immobilized Cells AndEnzymes” [IRL Press, (1986)]; B. Perbal, “A Practical Guide To MolecularCloning” (1984).

Therefore, if appearing herein, the following terms shall have thedefinitions set out below.

The terms “mammalian sterile 20-like kinase-1”, “Mst1” and any variantsnot specifically listed, may be used herein interchangeably, and as usedthroughout the present application and claims refer to proteinaceousmaterial including single or multiple proteins, and extends to thoseproteins having the profile of activities set forth herein and in theClaims. Accordingly, proteins displaying substantially equivalent oraltered activity are likewise contemplated. These modifications may bedeliberate, for example, such as modifications obtained through sitedirected mutagenesis, or may be accidental, such as those obtainedthrough mutations in hosts that are producers of the complex or itsnamed subunits. Also, the terms “mammalian sterile 20-like kinase-1”,“Mst1” and “Mst1(s)” are intended to include within their scope proteinsspecifically recited herein as well as all substantially homologousanalogs and allelic variations. The protein and encoding nucleic acidsequences of Mst1 are publicly known and described including in theGenbank database.

The amino acid residues described herein are preferred to be in the “L”isomeric form. However, residues in the “D” isomeric form can besubstituted for any L-amino acid residue, as long as the desiredfunctional property is retained by the polypeptide. NH₂ refers to thefree amino group present at the amino terminus of a polypeptide. COOHrefers to the free carboxy group present at the carboxy terminus of apolypeptide. In keeping with standard polypeptide nomenclature, J. Biol.Chem., 243:3552-59 (1969), abbreviations for amino acid residues (forexample for tyrosine, the 1-abbreviation Y, or the 3-letter abbreviationTyr) are as well known and recognized in the art.

A “replicon” is any genetic element (e.g., plasmid, chromosome, virus)that functions as an autonomous unit of DNA replication in vivo; i.e.,capable of replication under its own control.

A “vector” is a replicon, such as plasmid, phage or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment.

A “DNA molecule” refers to the polymeric form of deoxyribonucleotides(adenine, guanine, thymine, or cytosine) in its either single strandedform, or a double-stranded helix. This term refers only to the primaryand secondary structure of the molecule, and does not limit it to anyparticular tertiary forms. Thus, this term includes double-stranded DNAfound, inter alia, in linear DNA molecules (e.g., restrictionfragments), viruses, plasmids, and chromosomes. In discussing thestructure of particular double-stranded DNA molecules, sequences may bedescribed herein according to the normal convention of giving only thesequence in the 5′ to 3′ direction along the nontranscribed strand ofDNA (i.e., the strand having a sequence homologous to the mRNA).

A DNA “coding sequence” is a double-stranded DNA sequence which istranscribed and translated into a polypeptide in vivo when placed underthe control of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxyl) terminus. Acoding sequence can include, but is not limited to, prokaryoticsequences, cDNA from eukaryotic mRNA, genomic DNA sequences fromeukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. Apolyadenylation signal and transcription termination sequence willusually be located 3′ to the coding sequence. Transcriptional andtranslational control sequences are DNA regulatory sequences, such aspromoters, enhancers, polyadenylation signals, terminators, and thelike, that provide for the expression of a coding sequence in a hostcell.

An “expression control sequence” is a DNA sequence that controls andregulates the transcription and translation of another DNA sequence. Acoding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then translated intothe protein encoded by the coding sequence.

The term “oligonucleotide,” as used herein in referring to the probe ofthe present invention, is defined as a molecule comprised of two or moreribonucleotides, preferably more than three. Its exact size will dependupon many factors which, in turn, depend upon the ultimate function anduse of the oligonucleotide.

The term “primer” as used herein refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product, which is complementary to a nucleic acid strand, isinduced, i.e., in the presence of nucleotides and an inducing agent suchas a DNA polymerase and at a suitable temperature and pH. The primer maybe either single-stranded or double-stranded and must be sufficientlylong to prime the synthesis of the desired extension product in thepresence of the inducing agent. The exact length of the primer willdepend upon many factors, including temperature, source of primer anduse of the method. For example, for diagnostic applications, dependingon the complexity of the target sequence, the oligonucleotide primertypically contains 15-25 or more nucleotides, although it may containfewer nucleotides.

The primers or oligonucleotides herein are selected to be“substantially” complementary to different strands of a particulartarget DNA sequence. This means that the primers must be sufficientlycomplementary to hybridize with their respective strands. Therefore, theprimer sequence need not reflect the exact sequence of the template. Forexample, a non-complementary nucleotide fragment may be attached to the5′ end of the primer, with the remainder of the primer sequence beingcomplementary to the strand. Alternatively, non-complementary bases orlonger sequences can be interspersed into the primer, provided that theprimer sequence has sufficient complementarity with the sequence of thestrand to hybridize therewith and thereby form the template for thesynthesis of the extension product.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

A cell has been “transformed” by exogenous or heterologous DNA when suchDNA has been introduced inside the cell. The transforming DNA may or maynot be integrated (covalently linked) into chromosomal DNA making up thegenome of the cell. In prokaryotes, yeast, and mammalian cells forexample, the transforming DNA may be maintained on an episomal elementsuch as a plasmid. With respect to eukaryotic cells, a stablytransformed cell is one in which the transforming DNA has becomeintegrated into a chromosome so that it is inherited by daughter cellsthrough chromosome replication. This stability is demonstrated by theability of the eukaryotic cell to establish cell lines or clonescomprised of a population of daughter cells containing the transformingDNA. A “clone” is a population of cells derived from a single cell orcommon ancestor by mitosis. A “cell line” is a clone of a primary cellthat is capable of stable growth in vitro for many generations.

Two DNA sequences are “substantially homologous” when at least about 75%(preferably at least about 80%, and most preferably at least about 90 or95%) of the nucleotides match over the defined length of the DNAsequences. Sequences that are substantially homologous can be identifiedby comparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II,supra; Nucleic Acid Hybridization, supra. It should be appreciated thatalso within the scope of the present invention are DNA sequencesencoding which code for a having the same amino acid sequence as SEQ IDNO:, but which are degenerate to SEQ ID NO:. By “degenerate to” is meantthat a different three-letter codon is used to specify a particularamino acid. It is well known in the art that specific and particularcodons can be used interchangeably to code for each specific amino acid.

Mutations can be made in Mst1 or the compounds of the present inventionsuch that a particular codon is changed to a codon which codes for adifferent amino acid. Such a mutation is generally made by making thefewest nucleotide changes possible. A substitution mutation of this sortcan be made to change an amino acid in the resulting protein in anon-conservative manner (i.e., by changing the codon from an amino acidbelonging to a grouping of amino acids having a particular size orcharacteristic to an amino acid belonging to another grouping) or in aconservative manner (i.e., by changing the codon from an amino acidbelonging to a grouping of amino acids having a particular size orcharacteristic to an amino acid belonging to the same grouping). Such aconservative change generally leads to less change in the structure andfunction of the resulting protein. A non-conservative change is morelikely to alter the structure, activity or function of the resultingprotein. The present invention should be considered to include sequencescontaining conservative changes which do not significantly alter theactivity or binding characteristics of the resulting protein.

The following is one example of various groupings of amino acids:

Amino Acids with Nonpolar R Groups

Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine, TryptophanMethionine

Amino Acids with Uncharged Polar R Groups

Glycine, Serine, Threonine, Cysteine, Tyrosine, Asparagine, Glutamine

Amino Acids with Charged Polar R Groups (Negatively Charged at Ph 6.0)

Aspartic acid, Glutamic acid

Basic Amino Acids (Positively Charged at pH 6.0)

Lysine, Arginine, Histidine (at pH 6.0)

Another grouping may be those amino acids with phenyl groups:Phenylalanine, Tryptophan, Tyrosine

Another grouping may be according to molecular weight (i.e., size of Rgroups): Glycine 75 Alanine 89 Serine 105 Proline 115 Valine 117Threonine 119 Cysteine 121 Leucine 131 Isoleucine 131 Asparagine 132Aspartic acid 133 Glutamine 146 Lysine 146 Glutamic acid 147 Methionine149 Histidine (at pH 6.0) 155 Phenylalanine 165 Arginine 174 Tyrosine181 Tryptophan 204

Particularly preferred substitutions are:

Lys for Arg and vice versa such that a positive charge may bemaintained;

Glu for Asp and vice versa such that a negative charge may bemaintained;

Ser for Thr such that a free —OH can be maintained; and

Gln for Asn such that a free NH₂ can be maintained.

Amino acid substitutions may also be introduced to substitute an aminoacid with a particularly preferable property. For example, a Cys may beintroduced a potential site for disulfide bridges with another Cys. AHis may be introduced as a particularly “catalytic” site (i.e., His canact as an acid or base and is the most common amino acid in biochemicalcatalysis). Pro may be introduced because of its particularly planarstructure, which induces turns in the protein's structure.

Two amino acid sequences are “substantially homologous” when at leastabout 70% of the amino acid residues (preferably at least about 80%, andmost preferably at least about 90 or 95%) are identical, or representconservative substitutions.

A “heterologous” region of the DNA construct is an identifiable segmentof DNA within a larger DNA molecule that is not found in associationwith the larger molecule in nature. Thus, when the heterologous regionencodes a mammalian gene, the gene will usually be flanked by DNA thatdoes not flank the mammalian genomic DNA in the genome of the sourceorganism. Another example of a heterologous coding sequence is aconstruct where the coding sequence itself is not found in nature (e.g.,a cDNA where the genomic coding sequence contains introns, or syntheticsequences having codons different than the native gene). Allelicvariations or naturally-occurring mutational events do not give rise toa heterologous region of DNA as defined herein.

An “antibody” is any immunoglobulin, including antibodies and fragmentsthereof, that binds a specific epitope. The term encompasses polyclonal,monoclonal, and chimeric antibodies, the last mentioned described infurther detail in U.S. Pat. Nos. 4,816,397 and 4,816,567.

An “antibody combining site” is that structural portion of an antibodymolecule comprised of heavy and light chain variable and hypervariableregions that specifically binds antigen.

The phrase “antibody molecule” in its various grammatical forms as usedherein contemplates both an intact immunoglobulin molecule and animmunologically active portion of an immunoglobulin molecule.

Exemplary antibody molecules are intact immunoglobulin molecules,substantially intact immunoglobulin molecules and those portions of animmunoglobulin molecule that contains the paratope, including thoseportions known in the art as Fab, Fab′, F(ab′)₂ and F(v), which portionsare preferred for use in the therapeutic methods described herein.

The phrase “monoclonal antibody” in its various grammatical forms refersto an antibody having only one species of antibody combining sitecapable of immunoreacting with a particular antigen. A monoclonalantibody thus typically displays a single binding affinity for anyantigen with which it immunoreacts. A monoclonal antibody may thereforecontain an antibody molecule having a plurality of antibody combiningsites, each immunospecific for a different antigen; e.g., a bispecific(chimeric) monoclonal antibody.

“Heart failure” refers to the incapacity of the heart to assume a normalcontractile function.

“Treat and treatment” refer to both therapeutic treatment andprophylactic or preventative measures, wherein the object is to preventor slow down (lessen) an undesired physiological condition, disorder ordisease or obtain beneficial or desired clinical results. For purposesof this invention, beneficial or desired clinical results include, butare not limited to, alleviation of symptoms; diminishment of extent ofcondition, disorder or disease; stabilized (i.e. not worsening) state ofcondition, disorder or disease; delay or slowing of condition, disorder,or disease progression; amelioration of the condition, disorder ordisease state, remission (whether partial or total), whether detectableor undetectable; or enhancement or improvement of condition, disorder ordisease. Treatment includes eliciting a cellular response that isclinically significant, without excessive levels of side effects.Treatment also includes prolonging survival as compared to expectedsurvival if not receiving treatment.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like, when administered to a human.

“Pharmaceutically acceptable salts” includes salts of compounds derivedfrom the combination of a compound and an organic or inorganic acid.These compounds are useful in both free base and salt form. In practice,the use of the salt form amounts to use of the base form; both acid andbase addition salts are within the scope of the present invention.

“Pharmaceutically acceptable acid addition salt” refers to saltsretaining the biological effectiveness and properties of the free basesand which are not biologically or otherwise undesirable, formed withinorganic acids such as hydrochloric acid, hydrobromic acid, sulfuricacid, nitric acid, phosphoric acid and the like, and organic acids suchas acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaricacid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicyclic acid and the like.

“Pharmaceutically acceptable base addition salts” include those derivedfrom inorganic bases such as sodium, potassium, lithium, ammonium,calcium, magnesium, iron, zinc, copper, manganese, aluminum salts andthe like. Particularly preferred are the ammonium, potassium, sodium,calcium and magnesium salts. Salts derived from pharmaceuticallyacceptable organic nontoxic bases include salts of primary, secondary,and tertiary amines, substituted amines including naturally occurringsubstituted amines, cyclic amines and basic ion exchange resins, such asisopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine,dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine,methylglucamine, theobromine, purines, piperizine, piperidine,N-ethylpiperidine, polyamine resins and the like. Particularly preferredorganic nontoxic bases are isopropylamine, diethylamine, ethanolamine,trimethamine, dicyclohexylamine, choline, and caffeine.

“Parenteral” refers to introduction of the polypeptide by intravenous,intraarterial, intraperitoneal, intramuscular, intraventricular,intracranial, subcutaneous, subdermal, transvaginal, oral, nasal orrectal routes.

“Mammal” refers to any animal classified as a mammal, including humans,domestic and farm animals, and zoo, sports and pet companion animalssuch as a household pet and other domesticated animal such as, but notlimited to, cattle, sheep, ferrets, swine, horses, poultry, rabbits,goats, dogs, cats, and the like. Preferred companion animals are dogsand cats. Preferably, the mammal is human.

“Patient” refers to a mammal, preferably a human, in need of treatmentof a condition, disorder or disease.

“Inhibitor” includes but is not limited to, any suitable molecule,compound, protein or fragment thereof, nucleic acid, formulation orsubstance that can regulate Mst1 activity in such a way that Mst1 isdecreased or wherein the effects of Mst1 are blocked or altered. Theinhibitor includes an antagonist of Mst1, particularly an Mst1 mutant oraltered form that acts as a dominant negative. An inhibitor of the Mst1pathway includes any substance that alters the effect of Mst1 at thelevel downstream or upstream of Mst1. These include downstream proteinscapable of being modulated, including activated, by a signal from Mst1or which are more active on a signal from Mst1 or in the presence ofphosphorylated Mst1 or increased relative levels of cleaved Mst1. Mst1pathway further includes the upstream proteins, including particulary akinase(s), which are capable of phosphorylating amino acid(s) on Mst1 orinvolved in the signal which results in phosphorylated Mst1 oractivated, cleaved Mst1. Exemplary downstream proteins include but arenot limited to caspase and myelinbasic protein. The inhibitor caninclude, but is not limited to the specifically identified Caspase 3,Calyculin A and Chelerythrine. The inhibitor includes compounds whichblock Mst1 expression, including transcription or translation, such asantisense nucleic acids, siRNAs and ribozymes.

As used herein, “pg” means picogram, “ng” means nanogram, “ug” or “μg”mean microgram, “mg” means milligram, “ul” or “μl” mean microliter, “ml”means milliliter, “l” means liter.

A DNA sequence is “operatively linked” to an expression control sequencewhen the expression control sequence controls and regulates thetranscription and translation of that DNA sequence. The term“operatively linked” includes having an appropriate start signal (e.g.,ATG) in front of the DNA sequence to be expressed and maintaining thecorrect reading frame to permit expression of the DNA sequence under thecontrol of the expression control sequence and production of the desiredproduct encoded by the DNA sequence. If a gene that one desires toinsert into a recombinant DNA molecule does not contain an appropriatestart signal, such a start signal can be inserted in front of the gene.

The term “standard hybridization conditions” refers to salt andtemperature conditions substantially equivalent to 5×SSC and 65° C. forboth hybridization and wash. However, one skilled in the art willappreciate that such “standard hybridization conditions” are dependenton particular conditions including the concentration of sodium andmagnesium in the buffer, nucleotide sequence length and concentration,percent mismatch, percent formamide, and the like. Also important in thedetermination of “standard hybridization conditions” is whether the twosequences hybridizing are RNA-RNA, DNA-DNA or RNA-DNA. Such standardhybridization conditions are easily determined by one skilled in the artaccording to well known formulae, wherein hybridization is typically10-20^(N)C below the predicted or determined T_(m) with washes of higherstringency, if desired.

Screening Methods and Assays

Once a compound that binds to Mst1 or otherwise modulates the Mst1pathway is selected, it can be tested for its ability to modulatecardiac myocyte apoptosis or function. The effect of compounds oncardiac myocyte apoptosis can also be tested in vivo. Cardiac functionand output can be measured in vivo in animals by methods and assays wellknown in the art, including as described herein. In one such example ofthis assay, including as described herein, a myocardial infarction isgenerated in animal by ligation of the left coronary artery and theanimals are then observed for cardiac myopathy and cardiac function.

The function of cardiac myocytes and status of the Mst1 pathway may bedetermined by assessing the activation of intracellular proteinsindicative of apoptosis and/or Mst1 activation, particularly including,but not limited to, proteins or kinases such as caspase, Mst1 cleavagestatus, MAPK, JNK, etc. which may be substrates for Mst1 or activated ormore active in the presence of phosphorylated or cleaved Mst1, or activeon initiation of apoptosis. In addition, the activation of Mst1 orphosphorylation of Mst1 or its substrate, including MBP, may beassessed. Thus, following the incubation of cardiac myocytes or othercells expressing Mst1 with a test compound, the cells are lysed andtheir intracellular contents subjected to the appropriate tests, such asWestern blots, kinase assays, and electrophoretic mobility gel shiftassays (EMSAs).

In one embodiment, the modulation comprises phosphorylation ofintracellular proteins in the Mst1 pathway, and more preferably ofkinases, particularly including Mst1. The methods of the presentinvention may utilize any of the appropriate assays available in the artfor determining whether a kinase has been phosphorylated. Preferably,the assays used are Western blots or kinase assays.

In an embodiment, methods for screening cardiotherapeutic compoundsbased on their ability to activate phosphatase(s) (partially orcompletely) are also provided herein. The compounds to be screened mayinclude compounds that bind to Mst1 or dephopsphorylate Mst1, andmethods for selecting such compounds are described above. Thephosphatases inhibit the kinases specific for cardiac myocyte apoptosisand/or function, including Mst1 and proteins (kinases) in the Mst1pathway which may be substrates for Mst1 or activated or more active inthe presence of phosphorylated Mst1, e.g. caspase. Preferably, thephosphatases are Mst1 specific or Mst1 pathway specific. While not beingbound to a particular theory, this method is feasible for this purposedue to the fact that in some instances a kinase activity is tightlyregulated by its corresponding phosphatase. In case of ERK1/2, thephosphatases are known as the mitogen activated protein kinasesphosphatase-1,2,3 (MKP-1,2,3). These phosphatases belong to a family ofdual specificity phosphatases, which are responsible for the removal ofphosphate groups from the threonine and tyrosine residues on theircorresponding kinases (Camps et al., FASEB J., 14, pp. 6-16, 1999). Theprompt removal of phosphate groups by phosphatases ensures that kinaseactivation is short-lived and that the level of phosphorylation is lowin a resting cell. However, in order for the phosphatase to be activeand remove phosphate groups, it also needs to be phosphorylated.Therefore, activation of phosphatase activity results in inactivation ofkinase activity. The ability of the test compounds to activatephosphatase(s) can be determined by performing Western blots or kinaseassays. See above. For additional details on assessing phosphataseactivity, see Muda et al., J Biol. Chem., 273:9323-9329, 1998, and Campset al., Science 280:1262-1265, 1998. If the compound is determined topossess enhanced phosphatase activity, it can further be tested in oneof the cardiac myocyte apoptosis or in vitro or in vivo assays todetermine its effect on cardiac function and/or cardiomyopathy.

In a further embodiment of this invention, commercial test kits suitablefor use by a medical specialist may be prepared to determine thepresence or absence of or the amount of Mst1 or Mst1 activity in cells.Accordingly, one class of such kits will contain at least the labeledMst1 or its binding partner, for instance an antibody specific thereto,and directions, of course, depending upon the method selected, e.g.,competitive, sandwich, and the like, all and any of which are methodswell known to the skilled artisan. The kits may also contain peripheralreagents such as buffers, stabilizers, etc.

Accordingly, a test kit may be prepared for the demonstration of thepresence or capability of cells for Mst1 activity, comprising:

(a) a predetermined amount of at least one labeled immunochemicallyreactive component obtained by the direct or indirect attachment of Mst1or a specific binding partner thereto, to a detectable label;

(b) other reagents; and

(c) directions for use of said kit.

More specifically, the diagnostic test kit may comprise:

(a) a known amount of Mst1 as described above (or a binding partner)generally bound to a solid phase to form an immunosorbent, or in thealternative, bound to a suitable tag, or plural such end products, etc.(or their binding partners) one of each;

(b) if necessary, other reagents; and

(c) directions for use of said test kit.

In a further variation, the test kit may be prepared and used for thepurposes stated above, which operates according to a predeterminedprotocol (e.g. competitive, sandwich, double antibody, etc.), andcomprises:

(a) a labeled component which has been obtained by coupling Mst1 to adetectable label;

(b) one or more additional immunochemical reagents of which at least onereagent is a ligand or an immobilized ligand, which ligand is selectedfrom the group consisting of:

-   -   (i) a ligand capable of binding with the labeled component (a);    -   (ii) a ligand capable of binding with a binding partner of the        labeled component (a);    -   (iii) a ligand capable of binding with at least one of the        component(s) to be determined; and    -   (iv) a ligand capable of binding with at least one of the        binding partners of at least one of the component(s) to be        determined; and

(c) directions for the performance of a protocol for the detectionand/or determination of one or more components of an immunochemicalreaction between Mst1 and a specific binding partner thereto.

Compounds and Agents

Examples of agents, candidate compounds or test compounds include, butare not limited to, nucleic acids (e.g., DNA and RNA), carbohydrates,lipids, proteins, peptides, peptidomimetics, small molecules and otherdrugs. In one preferred aspect, agents can be obtained using any of thenumerous suitable approaches in combinatorial library methods known inthe art, including: biological libraries; spatially addressable parallelsolid phase or solution phase libraries; synthetic library methodsrequiring deconvolution; the “one-bead one-compound” library method; andsynthetic library methods using affinity chromatography selection. Thebiological library approach is limited to peptide libraries, while theother four approaches are applicable to peptide, non-peptide oligomer orsmall molecule libraries of compounds (Lam, 1997, Anticancer Drug Des.12:145; U.S. Pat. No. 5,738,996; and U.S. Pat. No. 5,807,683).

Phage display libraries may be used to screen potential Mst1 modulators.Their usefulness lies in the ability to screen, for example, a librarydisplaying a billion different compounds with only a modest investmentof time, money, and resources. For use of phage display libraries in ascreening process, see, for instance, Kay et al., Methods, 240-246,2001. An exemplary scheme for using phage display libraries to identifycompounds that bind or interact with Mst1 may be described as follows:initially, an aliquot of the library is introduced into microtiter platewells that have previously been coated with target protein, e.g. Mst1.After incubation (e.g. 2 hrs), the nonbinding phage are washed away, andthe bound phage are recovered by denaturing or destroying the targetwith exposure to harsh conditions such as, for instance pH 2, butleaving the phage intact. After transferring the phage to another tube,the conditions are neutralized, followed by infection of bacteria withthe phage and production of more phage particles. The amplified phageare then rescreened to complete one cycle of affinity selection. Afterthree or more rounds of screening, the phage are plated out such thatthere are individual plaques that can be further analyzed. For example,the conformation of binding activity of affinity-purified phage for Mst1may be obtained by performing ELISAs. One skilled in the art can easilyperform these experiments. In one aspect, an Mst1 molecule used for anyof the assays may be selected from a recombinant Mst1 protein, an Mst1fusion protein, an analog, derivative, or mimic thereof. In a preferredaspect, Mst1 is a recombinant Mst1 protein.

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al., 1993, Proc. Natl. Acad.Sci. USA 90:6909; Erb et al., 1994, Proc. Natl. Acad. Sci. USA 91:11422;Zuckermann et al., 1994, J. Med. Chem. 37:2678; Cho et al., 1993,Science 261:1303; Carrell et al., 1994, Angew. Chem. Int. Ed. Engl.33:2059; Carell et al., 1994, Angew. Chem. Int. Ed. Engl. 33:2061; andGallop et al., 1994, J. Med. Chem. 37:1233.

Libraries of compounds may be presented, e.g., presented in solution(e.g., Houghten, 1992, Bio/Techniques 13:412-421), or on beads (Lam,1991, Nature 354:82-84), chips (Fodor, 1993, Nature 364:555-556),bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698;5,403,484; and 5,223,409), plasmids (Cull et al., 1992, Proc. Natl.Acad. Sci. USA 89:1865-1869) or phage (Scott and Smith, 1990, Science249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al., 1990,Proc. Natl. Acad. Sci. USA 87:6378-6382; and Felici, 1991, J. Mol. Biol.222:301-310).

Antibodies, including polyclonal and monoclonal antibodies, particularlyanti-Mst1 antibodies and neutralizing antibodies may be useful ascompounds to modulate cardiac myocyte apoptosis and/or function. Also,antibodies including both polyclonal and monoclonal antibodies, anddrugs that modulate the activity of Mst1 and/or their subunits maypossess certain diagnostic applications and may for example, be utilizedfor the purpose of detecting and/or measuring conditions such as cardiacdisease, cardiomyopathy, or cardiac function. Mst1 or its subunits maybe used to produce both polyclonal and monoclonal antibodies tothemselves in a variety of cellular media, by known techniques such asthe hybridoma technique utilizing, for example, fused mouse spleenlymphocytes and myeloma cells. Likewise, small molecules that mimic orantagonize the activity(ies) of Mst1 may be discovered or synthesized,and may be used in diagnostic and/or therapeutic protocols.

The general methodology for making monoclonal antibodies by hybridomasis well known. Immortal, antibody-producing cell lines can also becreated by techniques other than fusion, such as direct transformationof B lymphocytes with oncogenic DNA, or transfection with Epstein-Barrvirus. See, e.g., M. Schreier et al., “Hybridoma Techniques” (1980);Hammerling et al., “Monoclonal Antibodies And T-cell Hybridomas” (1981);Kennett et al., “Monoclonal Antibodies” (1980); see also U.S. Pat. Nos.4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917;4,472,500; 4,491,632; 4,493,890.

Panels of monoclonal antibodies produced against Mst1 peptides can bescreened for various properties; i.e., isotype, epitope, affinity, etc.Of particular interest are monoclonal antibodies that neutralize theactivity of Mst1. Such monoclonals can be readily identified in Mst1activity assays, for instance in kinase assays using MBP as substrate.

Once an Mst1 modulating compound has been optimally designed, forexample as described above, substitutions may then be made in some ofits atoms or chemical groups in order to improve or modify its bindingproperties, or its pharmaceutical properties such as stability ortoxicity. Generally, initial substitutions are conservative, i.e., thereplacement group will have approximately the same size, shape,hydrophobicity and charge as the original group. One of skill in the artwill understand that substitutions known in the art to alterconformation should be avoided. Such altered chemical compounds may thenbe analyzed for efficiency of modulating Mst1, including in assays asdescribed herein.

This invention also encompasses prodrug derivatives of the compoundscontained herein. The term “prodrug” refers to a pharmacologicallyinactive derivative of a parent drug molecule that requiresbiotransformation, either spontaneous or enzymatic, within the organismto release the active drug. Prodrugs are variations or derivatives ofthe compounds of this invention which have groups cleavable undermetabolic conditions. Prodrugs become the compounds of the inventionwhich are pharmaceutically active in vivo, when they undergo solvolysisunder physiological conditions or undergo enzymatic degradation. Prodrugcompounds of this invention may be called single, double, triple etc.,depending on the number of biotransformation steps required to releasethe active drug within the organism, and indicating the number offunctionalities present in a precursor-type form. Prodrug forms oftenoffer advantages of solubility, tissue compatibility, or delayed releasein the mammalian organism (see, Bundgard, Design of Prodrugs, pp. 7-9,21-24, Elsevier, Amsterdam 1985 and Silverman, The Organic Chemistry ofDrug Design and Drug Action, pp. 352-401, Academic Press, San Diego,Calif., 1992). Prodrugs commonly known in the art include acidderivatives well known to practitioners of the art, such as, forexample, esters prepared by reaction of the parent acids with a suitablealcohol, or amides prepared by reaction of the parent acid compound withan amine, or basic groups reacted to form an acylated base derivative.Moreover, the prodrug derivatives of this invention may be combined withother features herein taught to enhance bioavailability. The preparationof pharmaceutically acceptable isomers, solvates or hydrates would beapparent to one of ordinary skill in the art.

Methods and Therapeutic and Pharmaceutical Compositions

The Mst1 modulators of the present invention, particularly. Mst1inhibitors, may be prepared in pharmaceutical compositions, with asuitable carrier and at a strength effective for administration byvarious means to a patient experiencing cardiac disease, includingcardiomyopathy or compromised cardiac function, for the treatment oramelioration thereof. A variety of administrative techniques may beutilized, among them parenteral techniques such as subcutaneous,intravenous and intraperitoneal injections, catheterizations and thelike. Average quantities of the modulators or their subunits may varyand in particular should be based upon the recommendations andprescription of a qualified physician or veterinarian.

A pharmaceutical composition useful in the present invention comprisesan Mst1 inhibitor (such as described above) and a pharmaceuticallyacceptable carrier, excipient, diluent and/or salt. Pharmaceuticallyacceptable carrier, diluent, excipient and/or salt means that thecarrier, diluent, excipient and/or salt must be compatible with theother ingredients of the formulation, does not adversely affect thetherapeutic benefit of the Mst1 inhibitor, and is not deleterious to therecipient thereof.

In a preferred embodiment of the invention, a method of treating orameliorating cardiac disease is provided by administering compositionscomprising compounds identified by the screening methods providedherein. The Mst1 modulating compositions of the present invention may beutilized by providing an effective amount of such compositions to asubject in need thereof.

The invention provides a method for treating cardiac disease in a mammalcomprising administering to said mammal an effective amount of an Mst1inhibitor. In a particular aspect, the invention further provides amethod for treating a cardiac disease or dysfunction selected from thegroup of congestive heart failure, cardiomyopathy, including ischemicand nonischemic cardiomyopathy, coronary artery disease, arrhythmias,fibrosis of the heart, valve defects, and atherosclerosis in a mammalcomprising administering to said mammal an effective amount of an Mst1inhibitor. In a still further aspect a method is provided for reducingthe risk of cardiomyopathy or dysfunction in a mammal after said mammalhas suffered a myocardial infarct or other coronary event wherein bloodflow to the heart is compromised or significantly reduced comprisingadministering to said mammal an effective amount of an Mst1 inhibitor orMst1 pathway inhibitor. In an additional aspect, the invention providesa method of cardioprotection, wherein an inhibitor of Mst1 isadministered in conjunction with or following therapy with a compound ordrug which is cardiotoxic or has potential to be cardiotoxic. In thismethod, an Mst1 modulator blocks or otherwise reduces the cardiotoxiceffects of a drug or compound. In a particular embodiment, the drug orcompound is a chemotherapeutic agent, particularly an anti-cancer agent,including but not limited to doxorubicin. In a further aspect,administration of the Mst1 modulator enables chemotherapeutic,particularly anti-cancer or anti-tumor compounds, treatment withoutsignificant cardiotoxic effects or permits prolonged chemotherapy oradministration of higher concentrations or amounts of a potentiallycardiotoxic drug.

In general, for use in treatment, the compounds of the invention may beused alone or in combination with other compositions for the treatmentof heart disease, including but not limited to congestive heart failure,cardiomyopathy, including ischemic and nonischemic cardiomyopathy,coronary artery disease, arrhythmias, fibrosis of the heart, valvedefects, and atherosclerosis. In particular, an effective amount of anMst1 inhibitor may be used in combination with one or more compound fortreatment of cardiac disease or of atherosclerosis, including but notlimited to a beta-blocker, nitrate, calcium channel antagonists,angiotensin-converting enzyme (ACE) inhibitors, an anti-platelet drug,diuretics, digoxin and antilipemic agents, agents which altercholesterol or lipid metabolism. Further examples of such ortreatmantilipemic agents include, but are not limited to the statins,such as Lovastatin (Mevacor®), Atorvastatin (Lipitor®), Simvastatin(Zocor®), Cerivastatin (Baycol®), Pravastatin (Pravacol®), Fluvastatin(Lescol®) and Rosuvastatin; as well as other classes of compoundsincluding cholesterol ester transfer protein inhibitors; cholesteroladsorption inhibitors (e.g., Ezetimide®); fibrates (e.g., Gemfibrozil®);nicotinic acid (e.g., niacin and analogs, Niaspan®); bile acid reuptakeinhibitors; and modulators of nuclear hormone receptors such as PPARa,PPARb, PPARg, LXRa, LXRb, FXR, RORa and SHP.

Administration of the compounds or pharmaceutical compositions thereoffor practicing the present invention can be by any method that deliversthe compounds systemically. These methods include oral routes,parenteral routes, intraduodenal routes, etc.

For topical applications, the compound or pharmaceutical compositionthereof can be formulated in a suitable ointment containing the activecomponent suspended or dissolved in one or more carriers. Carriers fortopical administration of the compounds of this invention include, butare not limited to, mineral oil, liquid petrolatum, white petrolatum,propylene glycol, polyoxyethylene, polyoxypropylene compound,emulsifying wax, sugars such as lactose and water. Alternatively, thepharmaceutical compositions can be formulated in a suitable lotion orcream containing the active components suspended or dissolved in one ormore pharmaceutically acceptable carriers. Suitable carriers include,but are not limited to, mineral oil, sorbitan monostearate, polysorbate60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcoholand water.

Depending on the particular condition, disorder or disease to betreated, additional therapeutic agents can be administered together withthe Mst1 inhibitor. Those additional agents can be administeredsequentially in any order, as part of a multiple dosage regimen, fromthe Mst1 inhibitor intermittent administration). Alternatively, thoseagents can be part of a single dosage form, mixed together with the Mst1inhibitor in a single composition (simultaneous or concurrentadministration).

For oral administration, a pharmaceutical composition useful in theinvention can take the form of solutions, suspensions, tablets, pills,capsules, powders, granules, semisolids, sustained release formulations,elixirs, aerosols, and the like. Tablets containing various excipientssuch as sodium citrate, calcium carbonate and calcium phosphate areemployed along with various disintegrants such as starch, preferablypotato or tapioca starch, and certain complex silicates, together withbinding agents such as polyvinylpyrrolidone, sucrose, gelatin andacacia. Additionally, lubricating agents such as magnesium stearate,sodium lauryl sulfate and talc are often very useful for tablettingpurposes. Solid compositions of a similar type are also employed asfillers in soft and hard-filled gelatin capsules; preferred materials inthis connection also include lactose or milk sugar as well as highmolecular weight polyethylene glycols. When aqueous suspensions and/orelixirs are desired for oral administration, the compounds of thisinvention can be combined with various sweetening agents, flavoringagents, coloring agents, emulsifying agents and/or suspending agents, aswell as such diluents as water, ethanol, propylene glycol, glycerin andvarious like combinations thereof.

The choice of formulation depends on various factors such as the mode ofdrug administration (e.g., for oral administration, formulations in theform of tablets, pills or capsules are preferred) and thebioavailability of the drug substance. Recently, pharmaceuticalformulations have been developed especially for drugs that show poorbioavailability based upon the principle that bioavailability can beincreased by increasing the surface area i.e., decreasing particle size.For example, U.S. Pat. No. 4,107,288 describes a pharmaceuticalformulation having particles in the size range from 10 to 1,000 nm inwhich the active material is supported on a crosslinked matrix ofmacromolecules: U.S. Pat. No. 5,145,684 describes the production of apharmaceutical formulation in which the drug substance is pulverized tonanoparticles (average particle size of 400 nm) in the presence of asurface modifier and then dispersed in a liquid medium to give apharmaceutical formulation that exhibits remarkably highbioavailability.

The term “parenteral” as used herein refers to modes of administration,which include intravenous, intramuscular, intraperitoneal, intrasternal,subcutaneous, intramedullary and intraarticular injection and infusion.A pharmaceutical composition for parenteral injection can comprisepharmaceutically acceptable sterile aqueous or nonaqueous solutions,dispersions, suspensions or emulsions as well as sterile powders forreconstitution into sterile injectable solutions or dispersions justprior to use. Aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal injection purposes. Inthis connection, the sterile aqueous media employed are all readilyobtainable by standard techniques well-known to those skilled in theart. Examples of suitable aqueous and nonaqueous carriers, diluents,solvents or vehicles include water, ethanol, polyols (such as glycerol,propylene glycol, polyethylene glycol, and the like),carboxymethylcellulose and suitable mixtures thereof, vegetable oils(such as olive oil), and injectable organic esters such as ethyl oleate.Proper fluidity can be maintained, for example, by the use of coatingmaterials such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and by the use of surfactants.

The pharmaceutical compositions useful in the present invention can alsocontain adjuvants such as, but not limited to, preservatives, wettingagents, emulsifying agents, and dispersing agents. Prevention of theaction of microorganisms can be ensured by the inclusion of variousantibacterial and antifungal agents, such as for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It can also bedesirable to include isotonic agents such as sugars, sodium chloride,and the like. Prolonged absorption of the injectable pharmaceutical formcan be brought about by the inclusion of agents that delay absorptionsuch as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of the drugs, it isdesirable to slow the absorption from subcutaneous or intramuscularinjection. This can be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, can depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe drug in biodegradable polymers such as polylactide, polyglycolide,and polylactide-polyglycolide. Depending upon the ratio of drug topolymer and the nature of the particular polymer employed, the rate ofdrug release can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions that are compatible with body tissues.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium just prior to use.

Administration by slow infusion is particularly useful when intrathecalor epidural routes are employed. A number of implantable or bodymountable pumps useful in delivering compound at a regulated rate areknown in the art. See, e.g., U.S. Pat. No. 4,619,652.

Suspensions, in addition to the active compounds, can contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar, and tragacanth, and mixturesthereof.

For purposes of transdermal (e.g., topical) administration, dilutesterile, aqueous or partially aqueous solutions (usually in about 0.1%to 5% concentration), otherwise similar to the above parenteralsolutions, are prepared.

The pharmaceutical compositions useful in the invention can also beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and can be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

In nonpressurized powder compositions, the active ingredients in finelydivided form can be used in admixture with a larger-sizedpharmaceutically acceptable inert carrier comprising particles having asize, for example, of up to 100 μm in diameter. Suitable inert carriersinclude sugars such as lactose. Desirably, at least 95% by weight of theparticles of the active ingredient have an effective particle size inthe range of 0.01 to 10 μm.

Alternatively, the composition can be pressurized and contain acompressed gas, such as, e.g., nitrogen, carbon dioxide or a liquefiedgas propellant. The liquefied propellant medium and indeed the totalcomposition are preferably such that the active ingredients do notdissolve therein to any substantial extent. The pressurized compositioncan also contain a surface active agent. The surface active agent can bea liquid or solid non-ionic surface active agent or can be a solidanionic surface active agent. It is preferred to use the solid anionicsurface active agent in the form of a sodium salt.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of theinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat room temperature but liquid at body temperature and therefore melt inthe rectum or vaginal cavity and release the drugs.

The compositions useful in the present invention can also beadministered in the form of liposomes. As is known in the art, liposomesare generally derived from phospholipids or other lipid substances.Liposomes are formed by mono- or multi-lamellar hydrated liquid crystalsthat are dispersed in an aqueous medium. Any non-toxic, physiologicallyacceptable and metabolizable lipid capable of forming liposomes can beused. The present compositions in liposome form can contain, in additionto the compounds of the invention, stabilizers, preservatives,excipients, and the like. The preferred lipids are the phospholipids andthe phosphatidyl cholines (lecithins), both natural and synthetic.Methods to form liposomes are known in the art (see e.g., Prescott, E.,Meth. Cell Biol. 14:33 (1976)).

Other pharmaceutically acceptable carrier includes, but is not limitedto, a non-toxic solid, semisolid or liquid filler, diluent,encapsulating material or formulation auxiliary of any type, includingbut not limited to ion exchangers, alumina, aluminum stearate, lecithin,serum proteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat.

Solid pharmaceutical excipients include, but are not limited to, starch,cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour,chalk, silica gel, magnesium stearate, sodium stearate, glycerolmonostearate, sodium chloride, dried skim milk and the like. Liquid andsemisolid excipients can be selected from glycerol, propylene glycol,water, ethanol and various oils, including those of petroleum, animal,vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineraloil, sesame oil, etc. Preferred liquid carriers, particularly forinjectable solutions, include water, saline, aqueous dextrose, andglycols.

Methods of preparing various pharmaceutical compositions with a certainamount of active ingredient are known, or will be apparent in light ofthis disclosure, to those skilled in this art. Other suitablepharmaceutical excipients and their formulations are described inRemington's Pharmaceutical Sciences, edited by E. W. Martin, MackPublishing Company, 19th ed. (1995).

Pharmaceutical compositions useful in the present invention can contain0.1%-95% of the compound(s) of this invention, preferably 1%-70%. In anyevent, the composition or formulation to be administered will contain aquantity of a compound(s) according to this invention in an amounteffective to treat the condition, disorder or disease of the subjectbeing treated.

One of ordinary skill in the art will appreciate that pharmaceuticallyeffective amounts of the Mst1 inhibitor can be determined empiricallyand can be employed in pure form or, where such forms exist, inpharmaceutically acceptable salt, ester or prodrug form. The agents canbe administered to a patient as pharmaceutical compositions incombination with one or more pharmaceutically acceptable excipients. Itwill be understood that, when administered to, for example, a humanpatient, the total daily usage of the agents or composition of thepresent invention will be decided within the scope of sound medicaljudgement by the attending physician. The specific therapeuticallyeffective dose level for any particular patient will depend upon avariety of factors: the type and degree of the cellular response to beachieved; activity of the specific agent or composition employed; thespecific agents or composition employed; the age, body weight, generalhealth, sex and diet of the patient; the time of administration, routeof administration, and rate of excretion of the agent; the duration ofthe treatment; drugs used in combination or coincidental with thespecific agent; and like factors well known in the medical arts. Forexample, it is well within the skill of the art to start doses of theagents at levels lower than those required to achieve the desiredtherapeutic effect and to gradually increase the dosages until thedesired effect is achieved.

For example, satisfactory results are obtained by oral administration ofthe compounds at dosages on the order of from 0.05 to 500 mg/kg/day,preferably 0.1 to 100 mg/kg/day, more preferably 1 to 50 mg/kg/day,administered once or, in divided doses, 2 to 4 times per day. Onadministration parenterally, for example, by i.v. bolus, drip orinfusion, dosages on the order of from 0.01 to 1000 mg/kg/day,preferably 0.05 to 500 mg/kg/day, and more preferably 0.1 to 100mg/kg/day, can be used. Suitable daily dosages for patients are thus onthe order of from 2.5 to 500 mg p.o., preferably 5 to 250 mg p.o., morepreferably 5 to 100 mg p.o., or on the order of from 0.5 to 250 mg i.v.,preferably 2.5 to 125 mg i.v. and more preferably 2.5 to 50 mg i.v.

Dosaging can also be arranged in a patient specific manner to provide apredetermined concentration of the agents in the blood, as determined bytechniques accepted and routine in the art (HPLC is preferred). Thuspatient dosaging can be adjusted to achieve regular on-going bloodlevels, as measured by HPLC, on the order of from 50 to 5000 ng/ml,preferably 100 to 2500 ng/ml.

The preparation of therapeutic compositions which contain polypeptides,analogs or active fragments as active ingredients is well understood inthe art. Typically, such compositions are prepared as injectables,either as liquid solutions or suspensions, however, solid forms suitablefor solution in, or suspension in, liquid prior to injection can also beprepared. The preparation can also be emulsified. The active therapeuticingredient is often mixed with excipients which are pharmaceuticallyacceptable and compatible with the active ingredient. Suitableexcipients are, for example, water, saline, dextrose, glycerol, ethanol,or the like and combinations thereof. In addition, if desired, thecomposition can contain minor amounts of auxiliary substances such aswetting or emulsifying agents, pH buffering agents which enhance theeffectiveness of the active ingredient.

A polypeptide, analog or active fragment can be formulated into thetherapeutic composition as neutralized pharmaceutically acceptable saltforms. Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide or antibodymolecule) and which are formed with inorganic acids such as, forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, oxalic, tartaric, mandelic, and the like. Salts formed from thefree carboxyl groups can also be derived from inorganic bases such as,for example, sodium, potassium, ammonium, calcium, or ferric hydroxides,and such organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

The therapeutic polypeptide-, analog-, -modulator or activefragment-containing compositions are conventionally administeredintravenously, as by injection of a unit dose, for example. The term“unit dose” when used in reference to a therapeutic composition of thepresent invention refers to physically discrete units suitable asunitary dosage for humans, each unit containing a predetermined quantityof active material calculated to produce the desired therapeutic effectin association with the required diluent; i.e., carrier, or vehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered depends on the subject to be treated, capacity of thesubject's immune system to utilize the active ingredient, and degree ofinhibition or neutralization of ˜binding capacity desired. Preciseamounts of active ingredient required to be administered depend on thejudgment of the practitioner and are peculiar to each individual.However, suitable dosages may range from about 0.1 to 20, preferablyabout 0.5 to about 10, and more preferably one to several, milligrams ofactive ingredient per kilogram body weight of individual per day anddepend on the route of administration. Suitable regimes for initialadministration and booster shots are also variable, but are typified byan initial administration followed by repeated doses at one or more hourintervals by a subsequent injection or other administration.Alternatively, continuous intravenous infusion sufficient to maintainconcentrations of ten nanomolar to ten micromolar in the blood arecontemplated.

The therapeutic compositions may further include an effective amount ofthe Mst1 antagonist or analog or modulator thereof, and one or more ofthe following active ingredients, including but not limited to: anantibiotic, a steroid, an anticoagulant, a statin or other cholesterollowering drug, an anti-hypertensive drug, an immune modulatory drug.

Another feature of this invention is the expression of the DNA sequencesdisclosed herein, including Mst1, particularly altered, includingdominant negative forms of Mst1. As is well known in the art, DNAsequences may be expressed by operatively linking them to an expressioncontrol sequence in an appropriate expression vector and employing thatexpression vector to transform an appropriate unicellular host.

Such operative linking of a DNA sequence of this invention to anexpression control sequence, of course, includes, if not already part ofthe DNA sequence, the provision of an initiation codon, ATG, in thecorrect reading frame upstream of the DNA sequence.

A wide variety of host/expression vector combinations may be employed inexpressing the DNA sequences of this invention. Useful expressionvectors, for example, may consist of segments of chromosomal,non-chromosomal and synthetic DNA sequences. Suitable vectors includederivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmidscol El, pCR1, pBR322, pMB9 and their derivatives, plasmids such as RP4;phage DNAS, e.g., the numerous derivatives of phage □, e.g., NM989, andother phage DNA, e.g., M13 and filamentous single stranded phage DNA;yeast plasmids such as the 2μ plasmid or derivatives thereof; vectorsuseful in eukaryotic cells, such as vectors useful in insect ormammalian cells; vectors derived from combinations of plasmids and phageDNAs, such as plasmids that have been modified to employ phage DNA orother expression control sequences; and the like.

Any of a wide variety of expression control sequences—sequences thatcontrol the expression of a DNA sequence operatively linked to it—may beused in these vectors to express the DNA sequences of this invention.Such useful expression control sequences include, for example, the earlyor late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lacsystem, the trp system, the TAC system, the TRC system, the LTR system,the major operator and promoter regions of phage □, the control regionsof fd coat protein, the promoter for 3-phosphoglycerate kinase or otherglycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5), thepromoters of the yeast-mating factors, and other sequences known tocontrol the expression of genes of prokaryotic or eukaryotic cells ortheir viruses, and various combinations thereof.

A wide variety of unicellular host cells are also useful in expressingthe DNA sequences of this invention. These hosts may include well knowneukaryotic and prokaryotic hosts, such as strains of E. coli,Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animalcells, such as CHO, R1.1, B—W and L-M cells, African Green Monkey kidneycells (e.g., COS 1, COS 7, BSC1, BSC40, and BMT10), insect cells (e.g.,Sf9), and human cells and plant cells in tissue culture.

It will be understood that not all vectors, expression control sequencesand hosts will function equally well to express the DNA-sequences ofthis invention. Neither will all hosts function equally well with thesame expression system. However, one skilled in the art will be able toselect the proper vectors, expression control sequences, and hostswithout undue experimentation to accomplish the desired expressionwithout departing from the scope of this invention. For example, inselecting a vector, the host must be considered because the vector mustfunction in it. The vector's copy number, the ability to control thatcopy number, and the expression of any other proteins encoded by thevector, such as antibiotic markers, will also be considered.

In selecting an expression control sequence, a variety of factors willnormally be considered. These include, for example, the relativestrength of the system, its controllability, and its compatibility withthe particular DNA sequence or gene to be expressed, particularly asregards potential secondary structures. Suitable hosts will be selectedby consideration of, e.g., their compatibility with the chosen vector,their secretion characteristics, their ability to fold proteinscorrectly, and their fermentation requirements, as well as the toxicityto the host of the product encoded by the DNA sequences to be expressed,and the ease of purification of the expression products.

Considering these and other factors a person skilled in the art will beable to construct a variety of vector/expression control sequence/hostcombinations that will enable expression the DNA sequences encoding Mst1or an antagonist of Mst1, including for example a mutant and dominantnegative Mst1, of this invention on transduction to animal cells, oninfection of animal cells, or on fermentation or in large scale animalculture.

It is further intended that Mst1 analogs may be prepared from nucleotidesequences of the protein complex/subunit derived within the scope of thepresent invention. Analogs, such as fragments, may be produced, forexample, by pepsin or other protease digestion of Mst1 polypeptide.Other analogs, such as muteins, can be produced by standardsite-directed mutagenesis of Mst1 coding sequences. Analogs exhibiting“Mst1 activity”, including kinase activity, such as small molecules,whether functioning as promoters or inhibitors, may be identified byknown in vivo and/or in vitro assays.

As mentioned above, a DNA sequence encoding Mst1 or an analog, includinga dominant negative mutant of Mst1, can be prepared synthetically ratherthan cloned. The DNA sequence can be designed with the appropriatecodons for the Mst1 amino acid sequence. In general, one will selectpreferred codons for the intended host if the sequence will be used forexpression. The complete sequence is assembled from overlappingoligonucleotides prepared by standard methods and assembled into acomplete coding sequence. See e.g., Edge, Nature, 292:756 (1981);Nambair et al., Science, 223:1299 (1984); Jay et al., J. Biol. Chem.,259:6311 (1984). Synthetic DNA sequences allow convenient constructionof genes which will express Mst1 analogs or “muteins”. Alternatively,DNA encoding muteins can be made by site-directed mutagenesis of nativeMst1 genes or cDNAs, and muteins can be made directly using conventionalpolypeptide synthesis. A general method for site-specific incorporationof unnatural amino acids into proteins is described in Christopher J.Noren, Spencer J. Anthony-Cahill, Michael C. Griffith, Peter G. Schultz,Science, 244:182-188 (April 1989). This method may be used to createanalogs with unnatural amino acids.

The present invention extends to the preparation of antisenseoligonucleotides, small interfering RNAs (siRNAs), and ribozymes thatmay be used to interfere with the expression of Mst1 at thetranslational level. This approach utilizes antisense nucleic acid andribozymes to block translation of a specific mRNA, either by maskingthat mRNA with an antisense nucleic acid, destabilizing it or cleavingit with a ribozyme.

Antisense nucleic acids are DNA or RNA molecules that are complementaryto at least a portion of a specific mRNA molecule. (See Weintraub, 1990;Marcus-Sekura, 1988.) In the cell, they hybridize to that mRNA, forminga double stranded molecule. The cell does not translate an mRNA in thisdouble-stranded form. Therefore, antisense nucleic acids interfere withthe expression of mRNA into protein. Oligomers of about fifteennucleotides and molecules that hybridize to the AUG initiation codonwill be particularly efficient, since they are easy to synthesize andare likely to pose fewer problems than larger molecules when introducingthem into Mst1-producing cells. Antisense methods have been used toinhibit the expression of many genes in vitro (Marcus-Sekura, 1988;Hambor et al., 1988).

Ribozymes are RNA molecules possessing the ability to specificallycleave other single stranded RNA molecules in a manner somewhatanalogous to DNA restriction endonucleases. Ribozymes were discoveredfrom the observation that certain mRNAs have the ability to excise theirown introns. By modifying the nucleotide sequence of these RNAs,researchers have been able to engineer molecules that recognize specificnucleotide sequences in an RNA molecule and cleave it (Cech, 1988.).Because they are sequence-specific, only mRNAs with particular sequencesare inactivated.

Investigators have identified two types of ribozymes, Tetrahymena-typeand “hammerhead”-type. (Hasselhoff and Gerlach, 1988) Tetrahymena-typeribozymes recognize four-base sequences, while “hammerhead”-typerecognize eleven- to eighteen-base sequences. The longer the recognitionsequence, the more likely it is to occur exclusively in the target mRNAspecies. Therefore, hammerhead-type ribozymes are preferable toTetrahymena-type ribozymes for inactivating a specific mRNA species, andeighteen base recognition sequences are preferable to shorterrecognition sequences.

The DNA sequences described herein may thus be used to prepare antisensemolecules against, and ribozymes that cleave mRNAs for Mst1.

The use of RNA interference strategies to inhibit the expression of Mst1is further embodied in the invention. Thus, methods of RNA interferenceand small interfering RNA compositions are included in the methods andcompositions of the present invention. RNA interference refers to thesilencing of genes specifically by double stranded RNA (dsRNA) (Fine, A.et al (1998) Nature 391; 806-811). In one embodiment, short or smallinterfering RNA (siRNA) is utilized (Elbashir, S. M. et al (2001) Nature411:494-498). In addition, long double stranded RNA hairpins may beemployed (Tavernarakis, N. et al (2000) Nature Genet. 24:180-183;Chuang, C. F. and Meyerowitz, E. M. (2000) PNAS USA 97:4985-90; Smith, NA et al (2000) Nature 407:319-20). Virus-mediated RNA interferenceagainst K-Ras has been described (B rummelkamp, T. R. et al (2002)Cancer Cell. 2:243-247).

The invention may be better understood by reference to the followingnon-limiting Examples, which are provided as exemplary of the invention.The following examples are presented in order to more fully illustratethe preferred embodiments of the invention and should in no way beconstrued, however, as limiting the broad scope of the invention.

EXAMPLE 1

The studies described herein were undertaken to determine whether or notMst1 plays an essential role in mediating apoptosis in cardiac myocytes,by using both cultured cardiac myocytes in vitro and transgenic mousemodels in vivo. The results demonstrate that Mst1 is activated not onlyby genotoxic compounds but also by clinically relevant pathologicinsults in the heart. Stimulation of Mst1 causes cardiac myocyteapoptosis and dilated cardiomyopathy without any compensatory cardiacmyocyte hypertrophy. Furthermore, specific inhibition of endogenous Mst1by dominant negative Mst1 inhibits cardiac myocyte apoptosis andmyocardial infarction in response to I/R. These results indicate thatMst1 plays an essential role in mediating apoptosis by clinicallyrelevant pathologic stimuli in the heart and identify Mst1 as animportant therapeutic target in ischemic heart diseases.

Mst1 is a Predominant MBP Kinase Activated by Potent ProapoptoticStimuli in Cardiac Myocytes

Our results suggest that Mst1 is a prominent MBP kinase activated bypotent apoptotic stimuli, including chelerythrine and calyculin A, incardiac myocytes. Activation of Mst 1 was also observed in response tohypoxia/reoxygenation of cardiac myocyte in vitro and I/R of the mouseheart in vivo. Activation of Mst1 by such clinically relevant stresseshas not been reported previously. Overexpression of wild type Mst1 wassufficient to cause typical findings of apoptosis, including cellshrinkage, cytoplasmic accumulation of mono- and oligo-nucleosomes andactivation of caspase-3 in cardiac myocytes. Cell death induced by wildtype Mst1 was suppressed in the presence of the caspase-3 inhibitor.These results suggest that activation of Mst1 activates caspase-3, whichin turn mediates cardiac myocyte apoptosis. Since cleavage andactivation of Mst1 were at least in part inhibited by the caspaseinhibitors, activation of Mst1 partially depends upon caspases. Thus,Mst1 and caspase-3 stimulate one another, thereby initiating a positivefeed back mechanism leading to increased apoptosis in cardiac myocytes.These results suggest that Mst1 can be an important regulator ofapoptosis in cardiac myocytes. This notion is also well supported by thefact that cardiac myocyte apoptosis by chelerythrine or calyculin Atreatment as well as in response to I/R was significantly suppressed inthe presence of dominant negative Mst1.

Recent evidence suggests that Mst1 is activated either byphosphorylation of the catalytic domain or by caspase-mediated cleavageof the C-terminal inhibitory domain (42). In fact, Mst1 is activated byboth mechanisms in cardiac myocytes: chelerythrine causes activation ofMst1 via caspase-dependent cleavage, while calyculin A causes activationof the full length form of Mst1 possibly through phosphorylation. Sinceboth mechanisms cause myocyte death, which was inhibited by dominantnegative Mst1, activation of Mst1by either mechanism causes cardiacmyocyte apoptosis. In this regard, it would be interesting to determine,by using a cleavage resistant form of Mst1, if the cleaved form and thefull-length form of Mst1 cause apoptosis with distinct morphologicalcharacteristics. Interestingly, the active form of Mst1 in Tg-Mst1 or incontrol mice subjected to I/R predominantly exists in the full lengthform. This indicates that cleavage of Mst1 by preceded activation ofcaspase-3 may not be required for activation of Mst1 in pathologicalconditions in vivo. Since partial cleavage of Mst1 was observed whenhigher doses of AdX-Mst1 were transduced in cardiac myocytes in vitro orI/R was applied to Tg-Mst1 (data not shown),we speculate that higherlevels of . . . caspase activation is required for the cleavage of Mst1to be seen. Since we could detect cleavage of caspase-3 even withoutcleavage of Mst1 in Tg-Mst1, caspase 3 may have a higher affinity forself-cleavage compared with cleavage of Mst1. Alternatively, activationof caspase-3 and cleavage of Mst1 could co-exist at the time only whencells are undergoing apoptosis, but anti-cleaved caspase-3 antibody maybe more sensitive for detection of the cleaved product.

The pattern of Mst1 activation by overexpression or by I/R is analogousto that by calyculin A, a phosphatase inhibitor. In this regard, it willbe interesting to examine if the activation loop of Mst1 isphosphorylated in Tg-Mst1 or in response to I/R (42). It has beenreported that transgenic mice overexpressing an inhibitor of PP-2Aexhibited dilated cardiomyopathy (43). It would be interesting toexamine if the activation of Mst1 is involved in development of heartfailure in these animals.

Cardiac Specific Overexpression of Mst1 Stimulates Cardiac MyocyteApoptosis and Induces Dilated Cardiomyopathy

Cardiac specific overexpression of wild type Mst1 caused dilation in all4 chambers, wall thinning, and reduced bi-ventricular function,consistent with findings of dilated cardiomyopathy. Interestingly,hypertrophy of surviving ventricular cardiac myocytes was not observed.To our knowledge, Mst1 is unique among protein kinases thus faroverexpressed in the mouse heart in vivo, because overexpression of Mst1in the heart primarily causes dilated cardiomyopathy without inducingcardiac myocyte hypertrophy. It should be noted that although cardiacfunction was generally reduced, some variability was observed in theseverity of congestive heart failure among transgenic littermate. Thissuggests that other genetic modifiers may exist to regulate thesusceptibility of mice to congestive heart failure.

We asked what is the cause of dilated cardiomyopathy. in Tg-Mst1l? Wefound that Tg-Mst1 have an increased number of TUNEL positive cells,which was accompanied by increased levels of caspase-3 activity.Although whether the TUNEL positive cells in Tg-Mst1 represent apoptosisor oncosis remains to be elucidated, since Mst1 promotes cell deathconsistent with apoptosis in vitro, it is likely that Tg-Mst1 mice havemore apoptosis. Considering the fact that increases in TUNEL positivemyocytes and interstitial fibrosis were observed even before the animalsmanifest overt sign of heart failure, increases in apoptosis may be theprimary cause of dilated cardiomyopathy. This notion is also supportedby the fact that contraction and relaxation function of single cardiacmyocytes isolated from Tg-Mst1 with decreased LVEF were preserved. Thus,we propose that decreases in the number of total cardiac myocytes in theheart (due to increases in apoptosis) at least in part may contribute toinitial development of dilated cardiomyopathy in Tg-Mst1.

It has been suggested that Mst1 is a MAP4K and regulates downstreamstress activated protein kinases, such as JNK and p38. The activatedform of Mst1 is translocated to the nucleus and potentially affectsnuclear components of the apoptosis machinery, including ICAD (Inhibitorof Caspase-Activated DNase) (31,32,42). However, the targets of Mst1mediating its proapoptotic effects in cardiac myocytes remain to beelucidated. Genomic analyses of Tg-Mst1 indicated that some componentsof nuclear encoded mitochondrial cytochrome c oxidase are downregulated.It has been shown that mitochondrial DNA encoded cytochrome c isdownregulated in the failing mouse heart possibly due to DNA damagecaused by increased levels of reactive oxygen species (44). It has beensuggested that downregulation of cytochrome c oxidase causes severeimpairment of mitochondrial ATP production as well as increasedproduction of reactive oxygen species, a potent inducer of apoptosis(45). It should be noted that reduction of mRNA of some components ofcytochrome c oxidase was observed in young Tg-Mst1 mice, which yetshowed overt sign of congestive heart failure. Thus, we propose thatdownregulation of cytochrome c oxidase is at least in part involved inthe pathogenesis of DCM in Tg-Mst1. It should be noted thatdownregulation of cytochrome c oxidase is not necessarily the commonfeature of congestive heart failure. Thus, this may be a uniquedownstream target of Mst1. The precise signaling mechanism connectingMst1 and reduced cytochrome c oxidase expression remains to beelucidated.

Our results suggest that Mst1 is a MAP4K and regulates downstream SRPKs,such as p38-MAPK and p46-JNKs in cardiac myocytes. Whether or notp38-MAPK and/or p46-JNKs mediate proapoptotic effects of Mst1 is ofgreat interest. The activated form of Mst1 is translocated to thenucleus and may affect Death-associated protein 4 (DAP4) (51) and thenuclear components of the apoptosis machinery, including ICAD (Inhibitorof Caspase-Activated DNase) (31, 32, 42). Although Mst1 efficientlyphosphorylates MBP at least in vitro, patho-physiologically relevantsubstrates of Mst1, mediating the proapoptotic effect of Mst1, remain tobe elucidated in cardiac myocytes.

Cardiac Specific Overexpression of Mst1 Prevents Compensatory CardiacMyocyte Hypertrophy and Potentially Obscures Operation of theFrank-Starling Mechanism in Individual Cardiac Myocytes

One of the most surprising findings in this study was that hypertrophyof surviving ventricular cardiac myocytes was not observed in Tg-Mst1.To our knowledge, Mst1 is unique among protein kinases thus faroverexpressed in the mouse heart in vivo, because overexpression of Mst1in the heart primarily stimulates cardiac myocyte death withoutcompensatory hypertrophy. Absence of cardiac hypertrophy not only leavesthe wall stress elevated but also fails to compensate for the loss ofcardiac mass caused by apoptosis in Tg-Mst1. Importantly, thelongitudinal length as well as the cell volume of isolated LV myocyteswere significantly smaller in Tg-Mst1. Although increases in myocytelength are commonly observed in dilated cardiomyopathy, to ourknowledge, decreases in cell volume or cardiac myocyte length in dilatedcardiomyopathy have not been previously reported. We speculate thatside-to-side slippage of cardiac myocytes (49), rather than elongationof individual myocytes, supports ventricular dilation in Tg-Mst1. Thus,increased tension development in dilated hearts according to theFrank-Starling would become less efficient at a single cell level inTg-Mst1, which may further contribute to decreased cardiac function inthese animals.

At present, we are not certain why compensatory cardiac myocytehypertrophy does not take place in Tg-Mst1 despite elevated wall stress.Mst1 may inhibit signaling molecules causing hypertrophy through eitherdirect phosphorylation or caspase-mediated cleavage. Little is known asto the cellular actions of Mst1 besides apoptosis. If Mst1 possess suchdirect anti-hypertrophic function, this would be a novel function ofMst1. Alternatively, ongoing cell proliferation could make cardiacmyocytes smaller (17). In this regard, whether or not increased celldeath stimulates myocyte proliferation in Tg-Mst1 remains to beelucidated.

Mst1 Plays an Important Role in Mediating Cardiac Myocyte Death inResponse to I/R in the Heart

Although endogenous Mst1 is activated by proapoptotic stimuli in bothneonatal rat cardiac myocytes and adult mouse heart, one may argue thatoverexpression of Mst1 causes unphysiological responses. In order toaddress this issue, we tested the effect of dominant negative Mst1(K59R) and confirmed that it was able to suppress the activity ofendogenous Mst1 in baseline and in response to I/R. Most importantly,cardiac specific expression of dominant negative Mst1 significantlyreduced the extent of myocardial infarction. Since increases in bothTUNEL positive cells and DNA laddering by I/R were significantlysuppressed in Tg-Mst1 (K59R), inhibition of endogenous Mst1 most likelyreduced cardiac myocyte apoptosis. We cannot completely exclude thepossibility that overexpression of Mst1 (K59R) may affect activities ofother protein kinases. However, since Mst1 is a predominant MBP kinaseactivated by I/R, the effect of the Mst1 (K59R) is most likely mediatedthrough-inhibition of Mst1 or that of the closely related Mst 1 family.Our results clearly indicate that inhibition of Mst1 effectively reducescardiac myocyte death by I/R. Mst1 and caspase stimulate one another,thereby forming a positive feedback loop. Thus, inhibiting the keymolecule facilitating this amplification loop would effectively blockpromotion of apoptosis. It has been recently shown that NORE, anoncatalytic polypeptide homologous to the putative tumor suppressorRASSF1, associates with Mst1 and the NORE-Mst1 complex mediatesRas-dependent apoptosis in fibroblasts (46). Thus, Mst1 may be involvedin a wide variety of extracellular stimuli as well as environmentalstresses. In this regard, Mst1 could be an important therapeutic targetin many cardiovascular diseases.

Mst1 is a Predominant MBP Kinase Activated by Potent Stimulators ofApoptosis in Cardiac Myocytes

In order to identify the signaling mechanism inducing apoptosis incardiac myocytes, cardiac myocytes were treated with chelerythrine, apotent inducer of apoptosis (35). The whole cell extracts prepared fromchelerythrine-treated myocytes were subjected to in gel kinase assays,using myelin basic protein as a substrate (35). We found that a 34 kDkinase is most prominently activated by chelerythrine at 6-30 μM, theconcentrations where apoptosis and activation of caspase-3 are observed(35). Activation of the 34 kD kinase was accompanied by disappearance ofa 61 kD kinase (FIG. 1A). Since it has been shown in other cell typesthat Mst1, a 61kD kinase, is cleaved by caspases and a resultant 34 kDfragment containing the N-terminal kinase domain becomes active, weexamined if the 61kD and 34 kD proteins are Mst1 in our experiments.Immune complex kinase assays, using specific anti-Mst1 antibody raisedagainst the N-terminal 15 amino acids of Mst1, indicated that the 34 kDform of Mst1 is activated by the chelerythrine treatment (FIG. 1B).Furthermore, immunoblot analyses, using specific anti-Mst1 antibodies,indicated that the 61kD form of Mst1 is downregulated, while the 34 kDform of Mst1 is concomitantly upregulated after chelerythrine treatment(FIG. 1C), consistent with the changes in activities of the 61 and 34 kDproteins in the in-gel kinase assays. In order to examine if Mst1 isactivated by a caspase-dependent cleavage in cardiac myocytes, wetransduced cardiac myocytes with XIAP, which we have shown inhibitsapoptosis and activation of caspase-3 in cardiac myocytes (35).Activation and cleavage of Mst1 was completely inhibited in the presenceof XIAP (FIG. 1B), suggesting that Mst1 is activated bycaspase-dependent cleavage in cardiac myocytes. Cleavage and activationof Mst-1 is also induced by hypoxia/reoxygenation, a known stimulus ofapoptosis in cardiac myocytes (FIG. 1D).

Recent evidence suggests that Mst1 is also activated by phosphorylationof the activation loop even in the absence of cleavage (42). CalyculinA, an inhibitor of protein phosphatase 2A (PP-2A) and PP-1, causesprominent cell death consistent with apoptosis in cardiac myocytes,which is evidenced by significant increases (4.3±1.4 fold, n=4, p<0.05vs untreated) in cytoplasmic accumulation of oligo- andmono-nucleosomes. In gel kinase assays indicated that a 61 kD form ofMst1 is strongly activated by calyculin A without activation of the 34kD form (FIG. 1E). These results suggest that Mst1 is a prominent MBPkinase, which is activated by potent apoptotic stimuli full length (61kD) forms, is accompanied by prominent cell death in cardiac myocytes.

EXAMPLE 2 Mst1 Plays an Essential Role in Mediating Apoptosis inResponse to Chelerythrine and Calyculin A in Cardiac Myocytes

In order to examine if Mst1 promotes apoptosis, we overexpressed eitherwild type Mst1 or dominant negative Mst1 (Mst1(K59R)) in cardiacmyocytes by using adenovirus transduction. Immunoblot analyses withanti-Mst1 antibody indicated that transduction of adenovirus harboringwild type Mst1 (AdX-Mst1) dose-dependently increased the full lengthform of Mst1. Expression of the cleaved form of Mst1 was also observedat high doses, suggesting that overexpression of Mst1 alone can inducepartial cleavage of Mst1 (FIG. 2A). By contrast, transduction ofadenovirus harboring Mst1(K59R) (AdX-DN-Mst1) increased only the fulllength form of Mst1(K59R) (FIG. 2B). In gel kinase assays showed thatthe MBP kinase activity of Mst1 was significantly increased byoverexpression of wild type Mst1, while it was not activated by Mst1(K59R), confirming that Mst1(K59R) is kinase inactive (FIG. 2C).Furthermore, increased activities and cleavage of Mst1 by overexpressionof Mst1 were attenuated in the presence of a caspase-3 inhibitor,DEVD-CHO, suggesting that Mst1 activates caspase-3, which in turn causescleavage of Mst1 (FIG. 2C, lane 4). Transduction of either control virusor AdX-DN-Mst1 did not induce any significant changes in the morphologyof cardiac myocytes. By contrast, transduction of AdX-Mst1 causedshrinkage and cell death in cardiac myocytes (FIG. 2D). Cytoplasmicaccumulation of mono- and oligo-nucleosomes, sensitive indicators ofnuclear fragmentation by apoptosis, was dose-dependently increased byoverexpression of wild type Mst1 but not by control virus or Mst1 (K59R)(FIG. 2E). Increases in DNA fragmentation by wild type Mst1 wereinhibited in the presence of DEVD-CHO (FIG. 2F). Overexpression of wildtype Mst1, but not Mst1 (K59R), caused activation of caspase-31 whichwas determined by immunoblotting with anti-cleaved specific caspase-3antibody (FIG. 2G). These results suggest that Mst1 induces cardiacmyocyte apoptosis in its kinase activity-dependent manner.

In order to examine if Mst1 is required for cardiac myocyte apoptosis inresponse to chelerythrine, cardiac myocytes were transduced withAdX-DN-Mst1 or control virus and challenged by chelerythrine.Cytoplasmic accumulation of mono- and oligo-nucleosome by chelerythrinetreatment was significantly suppressed in the presence of AdX-Mst1(K59R)but not by control virus (FIG. 3). DNA fragmentation by calyculin A wasalso inhibited by AdX-Mst1 (K59R) (5.6 fold with control virus vs 0.9fold with AdX-DN-Mst1. These results suggest that both cleaved and fulllength forms of Mst1 plays a critical role in mediating apoptosis incardiac myocytes.

EXAMPLE 3 Cardiac Specific Overexpression of Mst1 in Mice Causes DilatedCardiomyopathy Without Cardiac Myocyte Hypertrophy

In order to examine the function of Mst1 in the mouse heart in vivo,transgenic mice with cardiac specific overexpression of wild type(Tg-Mst1) was generated by using the □MHC promoter. We identified threefounders with transgene positive by Southern blot analyses. Among them,germ line transmission was observed in two lines. One line (line #28)showed prominent cardiac specific overexpression of wild type Mst1,while the other line (line #8) showed modest overexpression (FIG. 4A).We confirmed that Mst1 is overexpressed predominantly in the heart asexpected (FIG. 4B). In-gel MBP kinase assays indicated that the totalactivity of Mst1 is significantly increased in Tg-Mst1. Increases in theMBP kinase activity in Tg-Mst1 were found predominantly in the fulllength form rather than in the cleaved form (FIG. 4C).

Interestingly, some Tg-Mst1 exhibited overt signs of heart failure, suchas shortness of breath and edema, and died prematurely as early as onDay 15. Echocardiographic measurements of Tg-Mst1 lines #28 and #8 at70-80 days old indicated that Tg-Mst1 exhibited significant increases inLVEDD and LVESD and significant decreases in LVEF, LV fractionalshortening and LV wall thickness (TABLE 1). TABLE 1 EchocardiographicAnalyses of Tg-Mst1 Non-transgenic Tg-Mst1 (n = 4) (n = 5) Line #28(High level overexpression) Age (days old)   75 ± 2.89  76 ± 2.45 LVEDD(mm) 3.92 ± 0.11 4.39 ± 0.06* LVESD (mm) 2.62 ± 0.11 3.55 ± 0.10* LVEF(%) 70.0 ± 1.83 47.2 ± 2.80* % FS (%) 33.1 ± 1.44 19.2 ± 1.41* DSEP WT(mm) 0.59 ± 0.04 0.39 ± 0.01* DPW WT (mm) 0.59 ± 0.02 0.37 ± 0.03* HR(bpm) 246.8 ± 23.4  303.0 ± 32.8  *p < 0.01 compared with non-transgenicLine #8 (Intermediate level overexpression) Age (days old)  195 ± 1.96193 ± 1.96  LVEDD (mm) 4.40 ± 0.25 4.54 ± 0.28  LVESD (mm) 2.99 ± 0.143.29 ± 0.19  LVEF (%) 68.4 ± 1.21 61.6 ± 2.38* % FS (%) 31.9 ± 0.78 27.5± 1.46* DSEP WT (mm) 0.68 ± 0.02 0.64 ± 0.03  DPW WT (mm) 0.65 ± 0.020.63 ± 0.02  HR (bpm) 258.2 ± 8.1  255.8 ± 25.2  *p < 0.05 compared withnon-transgenic

Hemodynamic analyses conducted in Tg-Mst1 with (echocardiographicallydetermined) reduced LVEF confirmed that LVEDP was significantlyelevated, while LV dP/dt was decreased compared with non-transgeniclittermates (TABLE 2). TABLE 2 Hemodynamic Measurements of Tg-Mst1Non-transgenic (n = 4) Tg-Mst1 (n = 5) Age (days)  80 ± 7.8 88 ± 12 LVSP(mmHg)  84 ± 2.3  70 ± 5.1 LVEDP (mmHg) 3.3 ± 2.9  18.6 ± 1.5** +LVdP/dt 5200 ± 245  4300 ± 663  −LV dP/dt 4467 ± 356  3080 ± 481  RVSP(mmHg)  18 ± 2.9   29 ± 3.3* RVEDP (mmHg) 0.7 ± 0.4  9.0 ± 2.2* RAP(mmHg) 1.3 ± 0.4 7.0 ± 2.3 HR (bpm) 323 ± 54  297 ± 36 *p < 0.05,**p < 0.001 compared with non-transgenic

Necropsy of Tg-Mst1 performed at 1-2 months indicated dilation of all 4cardiac chambers, mural thrombus formation with occasional fibrousstructures in both atriums, and reduced wall thickness, consistent withthe findings of dilated cardiomyopathy (TABLE 3 and FIG. 4D). AlthoughLA and RA weight/body weight of Tg-Mst1 was significantly higher thanthat of non-transgenic littermate, LV weight/body weight and RVweight/body weight were not significantly different between Tg-Mst1 andnon-transgenic littermate (TABLE 3). Significant increases in lungweight/body weight and liver weight/body weight and visible congestionof the liver were observed in Tg-Mst1 compared with non-transgeniclittermate (TABLE 3 and FIG. 4E). The lungs were congested (FIG. 4F).Histological analyses of the heart sections indicated that TUNELpositive myocytes are significantly increased in Tg-Mst1 (control0.06±0.03%, Tg-Mst1 0.30±0.05%, n=4, p<0.01). The level of interstitialfibrosis significantly increased in all four chambers of Tg-Mst1 (FIG.4G). These results suggest that myocyte death and subsequent replacementof myocardium with fibrous tissue took place in Tg-Mst1 hearts.Interestingly, the cross sectional areas of cardiac myocytes in the LVwas not significantly different between Tg-Mst1 and non-transgenic miceconfirming that there was no compensatory cardiac myocyte hypertrophy inTg-Mst1 (FIG. 4H). The level of-cleaved caspase-3 was-increased inhearts of Tg-Mst1 (FIG. 4I). Although it was smaller in extent, reducedcardiac function and increased levels of cardiac myocyte apoptosis werealso found in line #8 of Tg-Mst1. TABLE 3 Postmortem PathologicMeasurements of Tg-Mst1 Non-transgenic Tg-Mst1 (n = 9) (n = 12) Age(days) 46.7 ± 2.7 46.9 ± 1.9 Body weight (g) 18.4 ± 0.4 19.4 ± 0.8 LV(mg) 64.2 ± 2.0 64.5 ± 2.6 RV (mg) 19.1 ± 0.8 20.6 ± 0.6 LV/body weight 3.48 ± 0.07  3.32 ± 0.06 Lung/body weight  6.67 ± 0.30  7.73 ± 0.29*Liver/body weight  47.6 ± 1.78  55.1 ± 2.71**p < 0.05 compared with non-transgenic**p < 0.01 compared with non-transgenic

EXAMPLE 4 Contractile Function of Individual Cardiac Myocytes Isolatedfrom Tg-Mst1 is not Altered

In order to examine the mechanism of reduced cardiac function inTg-Mst1, cardiac myocytes were isolated from 3 months old Tg-Mst1 andnon-transgenic control mice and their contractile properties wereevaluated by using the edge detection method. Echo cardiographicmeasurements indicated that LVEF of the Tg-Mst1 mice used in theseexperiments was significantly lower than that of non-transgenic mice(Tg-Mst1 51±5% vs non-transgenic littermate 68±1%, p<0.05, n=4). Therewere no significant differences in % contraction and the rate ofrelaxation of isolated cardiac myocytes between Tg-Mst1 andnon-transgenic mice (FIG. 5), suggesting that the decreases in cardiaccontractility in Tg-Mst1 may not be primarily caused by decreases in thecontractile or relaxation function of individual- cardiac myocytes.Interestingly, the length of ventricular myocytes isolated from Tg-Mst1was smaller than those from non-transgenic controls.

EXAMPLE 5 Expression of Nuclear Encoded Cytochrome c Oxidase is Reducedin Tg-Mst1

In order to examine the mechanism of dilated cardiomyopathy in Tg-Mst1,we performed DNA microarray analyses. PolyA RNA was isolated from thehearts of Tg-Mst1 and non-transgenic littermate at the age of 2 months,those who did not show overt signs of congestive heart failure. The listof the genes, whose expression was consistently upregulated ordownregulated in three independent analyses, is shown in TABLE 4.Classification of the identified genes according to their functionindicates that many genes regulating both mitochondrial and cytoplasmicmetabolism are downregulated in Tg-Mst1. Among them, we found that somecomponents of nuclear encoded cytochrome c oxidase subunits areconsistently downregulated by 21-64%. Exemplary RT-PCR results are shownin FIG. 8 which demonstrates that mRNA expression of PGC-1α and PGC-1βare each decreased in transgenic Tg-Mst1 mice. PGC-1 is a nucleartranscription co-factor which plays an important role in regulatingexpression of nuclear encoded mitochondrial genes. Proteomic analysis,although more limited in scope, showed similar results. In particularand exemplary of the protein analysis, FIG. 9 shows Western blots andtabulated expression of Cytochrome C oxidase complex proteins COX VIIa,COX IV and COX Vb in transgenic versus non-transgenic animals. TABLE 4Genes consistently upregulated or downregulated in Tg-Mst1 in threeindependent microarray analyses mean % change SEM Upregulated genesmatrix gamma carboxylglutamate protein 584 270 procollagen 1 alpha 1subunit 702 457 peptidylprolyl isomerase A 77 12 natriuretic peptideprecursor type B 819 278 desmin 199 95 Downregulated genes Mitochondrialmetabolism COX7A1 −74 15 COX7C −59 11 COX4 −47 20 COX5B −55 12 COX8B −5418 ATPase inhibitor −12 4 mitochondrial malate dehydrogenase −53 10soluble malate dehydrogenase −43 14 NADH dehydrogenase flavoprotein 1−52 2 NADH-ubiquinone oxidoreductase MWFE −54 24 subunit Cytoplasmicmetabolism gamma neuronal enolase 2 −62 8 muscle creatine kinase −72 4lactate dehydrogenase 2B subunit −40 8 soluble superoxide dismutase 1−28 4 prosaposin −85 10 Others vascular endothelial growth factor B −478 cardiac troponin I −59 11 la-associated invariant chain −64 25

EXAMPLE 6 Cardiac Specific Overexpression of Mst1 (K59R) Works asDominant Negative Mst1 and Significantly Reduces Myocardial Infarctionand Apoptosis in Response to I/R

In order to examine if Mst1 plays a critical role in mediating apoptosisin response to pathologic insults to the hearts, we generated transgenicmice with cardiac specific overexpression of dominant negative Mst1(Mst1 (K59R)) (Tg-DN-Mst1). Among three lines generated, line #10expressed the highest level of Mst1(K59R) in the heart (FIG. 6A).Tg-DN-Mst1 did not show premature death or any signs of heart failure.The results of echocardiographic measurements of Tg-DN-Mst1 were notsignificantly different from those of non-transgenic littermates (TABLE5). These results suggest that development of dilated cardiomyopathy inTg-Mst1 is dependent upon the kinase activity of Mst1. In order toexamine if overexpression of Mst1(K59R) works as dominant negative, weapplied 20 min of ischemia and subsequent 24h of reperfusion into themouse heart. Results of the in gel MBP kinase assays showed that I/Ractivates Mst1 primarily in the full length form in the control mousehearts (FIG. 6B). By contrast, activities of Mst1 at basal conditions aswell as in response to I/R were abolished in Tg-DN-Mst1, suggesting thatcardiac specific overexpression of Mst1(K59R) works as dominant negativefor endogenous Mst1 (FIG. 6C). The extent of myocardial infarction inresponse to I/R, quantitated by the TTC staining of the area at risk,was significantly smaller in Tg-DN-Mst1 compared with that innon-transgenic littermate. The heart section obtained from the ischemicarea of Tg-DN-Mst1 exhibited a smaller number of TUNEL positive myocytescompared with that of non-transgenic littermate (FIG. 6D).Electrophoresis of genomic DNA prepared from the ischemic area ofnon-transgenic mice exhibited a typical pattern of DNA laddering. Bycontrast, DNA laddering was significantly suppressed in Tg-DN-Mst1 (FIG.6E). These results suggest that inhibition of Mst1 reduces both cardiacmyocyte apoptosis and the size of myocardial infarction in mouse hearts.TABLE 5 Echocardiographic Analyses of Tg-DN-Mst1 NTg (n = 9) TG-DN-Mst1(n = 8) Age (days) 92 ± 5.5  102 ± 4.7   BW (g) 23 ± 0.6  22 ± 1.0  DSWT(mm) 0.6 ± 0.03 0.6 ± 0.02 LVDD (mm) 3.6 ± 0.11 3.9 ± 0.08 DPWT (mm) 0.6± 0.03 0.6 ± 0.03 LVSD (mm) 88 ± 6  87 ± 5  % EF (%) 68 ± 1.2  70 ± 0.9 % FS (%) 32 ± 0.9  33 ± 0.6  HR (bpm) 271 ± 16  231 ± 26 

EXAMPLE 7

We further examined the mechanism by which Mst1 stimulates apoptosis incardiac myocytes. Transduction of Mst1 (Tg-Mst1) significantly (twofold, n=3, P<0.05) increased the amount of cytochrome c in themitochondria-free cytosolic fraction compared with that of controlvirus, suggesting that release of cytochrome c may contribute to theproapoptotic effect of Mst1 (FIG. 7A). Transduction of Mst1 modestlyactivated p38-MAPK and p46-JNKs (FIGS. 7B and 7C), while that of thedominant negative Mst1 (Tg-DN-Mst1) abolished chelerythrine-inducedactivation of p38-MAPK in cardiac myocytes (FIG. 7D). These resultssuggest that Mst1 works as MAP4K in cardiac myocytes and that it playsan important role in mediating activation of p38-MAPK by chelerythrine.

EXAMPLE 8 Mst1 Directly Inhibits Cardiac Hypertrophy

We have previously and above shown that mammalian sterile-20 like kinase1 (Mst1) plays an important role in mediating cardiac myocyte apoptosisin response to ischemia/reperfusion and doxorubicin treatment. Cardiacspecific overexpression of Mst1 in transgenic mice (Tg-Mst1) increasescardiac myocyte apoptosis in the heart and the mice develop dilatedcardiomyopathy (DCM).and congestive heart failure within 3 months ofage. Surprisingly, histological analyses of the left ventricular (LV)cardiac myocyte cross sectional area and measurements of thelongitudinal length of isolated ventricular cardiac myocytes indicatedthat there was no compensatory cardiac myocyte hypertrophy in Tg-Mst1and that myocytes are actually shorter despite elevated wall stresscaused by DCM. Lack of compensatory cardiac myocyte hypertrophy mayadversely affect LV function of Tg-Mst1 because operation of the FrankStarling mechanism could be less efficient at the individual cell level.This study was undertaken in order to elucidate if transient expressionof Mst1 has a direct anti-hypertrophic effects in cultured cardiacmyocytes in vitro. Neonatal rat cardiac myocytes were transduced withadenoviral vectors (10 MOI) in serum-free conditions for 48 h and thentreated with phenylephrine (PE, 10 μM) for 48 h. Phenylephrinestimulates hypertrophy of cardiac myocytes in normal animals. Treatmentof LacZ virus-transduced myocytes with PE caused a 2.4 fold increase inthe cell size and a 1.7 fold increase in protein/DNA content. Bycontrast, transduction of adenovirus harboring Mst1 completely inhibitedPE-induced increases in cell size and protein/DNA content. Mst1 alsoinhibited PE-induced increases in mRNA expression of atrial natriureticfactor by 49%. Interestingly, Mst1 inhibited PE-induced (10 mintreatment) activation of ERK by 28%. In summary, Mst1 has a directanti-hypertrophic effect upon PE-treated cultured cardiac myocytes,possibly affecting other signaling mechanisms, including ERKs. Ourresults suggest that the proapoptotic signaling mechanism can directlyaffect hypertrophic signaling mechanisms, thereby contributing to thepathogenesis of cardiomyopathy.

EXAMPLE 9 Mst1 Plays an Essential Role in Mediating Cardiac MyocyteApoptosis by Doxorubicin and Doxorubicin-Induced Cardio-Toxicity

Mst1 is an ubiquitously expressed serine-threonine kinase, which isactivated by caspase-mediated cleavage and promotes apoptosis in manycell types, including cardiac myocytes. Doxorubicin (Dox or Doxo)belongs to the anthracycline class of anticancer drugs. The limitingfactor in the clinical use of Dox is the fact that it inducesdose-dependent and irreversible cardiomyopathy. Recent evidence suggeststhat Dox induces cardiac myocyte apoptosis, which may contribute to thecardiotoxicity of this compound. However, the signaling mechanism ofcardiac myocyte apoptosis by Dox is not well understood. We hypothesizedthat Mst1 mediates cardiac myocyte apoptosis by Dox. In order to addressthis issue, we used neonatal rat cardiac myocyte cultures and adenovirustransduction. Treatment of cardiac myocytes with Dox (0.1-3 μM)dose-dependently induced cell shrinkage within 6h (FIG. 10), which wasaccompanied by activation of caspase-3 and DNA fragmentation, which wasdetermined by cytoplasmic accumulation of mono- and oligo-nucleosome(FIGS. 11 and 12). These results suggest that Dox induces apoptosis incardiac myocytes. Immunoblot analyses indicated that Mst1 is cleaved toform a 34 kD fragment after 6h treatment with Dox at 0.1-3 μM (FIG. 16).Immunoblot analysis also demonstrated cleavage of caspase-3 andcaspase-9, to activate these enzymes in a time dependent manner ontreatment with Dox (FIGS. 13 and 14). In-gel kinase assays, using myelinbasic protein as a substrate, showed that cleavage of Mst1 isaccompanied by activation of Mst1 (FIG. 15). Interestingly,adenovirus-mediated transduction of dominant negative Mst1 significantlyreduced cell shrinkage and DNA fragmentation by Dox treatment, whilethat of control virus showed no effects (OD405 values after 6h treatmentwith 0.5 μM Dox: control virus 0.69 vs dominant negative Mst1 virus0.21) (FIG. 18). Dominant negative Mst1 mice receiving Dox maintainejection fraction and % functional shortening when compared to theirnon-transgenic littermates (FIGS. 19 and 20). In summary, Mst1 isactivated by Dox in cardiac myocytes. Mst1 plays an important role inmediating Dox-induced cardiac myocyte apoptosis. Mst1 may be animportant therapeutic target of Dox-induced cardiomyopathy.

EXAMPLE 10 Mst1 Plays a Critical Role in Cardiac Remodeling

Overexpression of dominant negative Mst1 (Tg-DN-Mst1) inhibits cardiacdilation and left ventricular dysfunction after myocardial infarction.Myocardial infarction was generated by ligation of the left coronaryartery in vivo in normal and Tg-DN-Mst1 transgenic mice. The results aredepicted in FIG. 23. FIG. 23A (left panel) indicates that a similar sizeof myocardial infarction was created by ligation of the left coronaryartery in non-transgenic (NTG) and transgenic (Tg-DN-Mst1) mice. LVEDD(FIG. 23B) and LVEF (FIG. 23C) were measured in sham operated and MImice. The results suggest that dominant negative Mst1 may preventcardiac dysfunction in post-MI patients.

Materials and Methods

Materials

Chelerythrine chloride, calyculin A and a caspase-3 inhibitor DEVD-CHOwere purchased from Biomol (Plymouth Meeting, Pa.).

Primary Culture of Neonatal Rat Ventricular Myocytes

Primary cultures of ventricular cardiac myocytes were prepared from1-day-old Crl: (WI) BR-Wistar rats (Charles River Laboratories,Wilmington, Mass.) as described previously (34). A cardiac myocyte richfraction was obtained by centrifugation through the discontinuousPercoll gradient as described (34). Cells were cultured in the cardiacmyocyte culture medium containing Dulbecco's modified Eagle medium(DMEM)/F12 supplemented with 5% horse serum, 4 μg/mL transferrin, 0.7ng/mL sodium selenite (GIBCO), 2 g/L bovine serum albumin (fraction V),3 mmol/L pyruvic acid, 15 mmol/L HEPES, 100 μmol/L ascorbic acid, 100μg/mL ampicillin, 5 μg/mL linoleic acid and 100 μmol/L5-bromo-2′-deoxyuridine (Sigma). We obtained myocyte cultures in whichmore than 95% were myocytes, as assessed by immunofluorescence stainingwith a monoclonal antibody against sarcomeric myosin (MF20). Culturemedia were changed to serum-free at 24-36 h and then subjected toadenovirus transduction. Myocytes were cultured in the serum-freecondition for 48 h before experiments.

Construction of the Adenoviral Vectors

We constructed recombinant adenovirus by using an Adeno-X adenovirusconstruction kit according to the manufacturer's instruction (Clontech,Palo Alto, Calif.). We made replication defective human adenovirus type5 (devoid of E1 and E3) harboring wild type Mst1 (AdX-Mst1) and dominantnegative Mst1 (AdX-Mst1(K59R)). Adenovirus harboring β-galactosidase(Ad5 βgal) was used as a control. Generation of adenovirus harboringXIAP (Ad5 XIAP) has been described (35). The method of adenovirustransduction has been described (35).

Analysis of DNA Fragmentation by ELISA and DNA Laddering Assays

Histone-associated DNA fragments were quantified by the Cell DeathDetection ELISA (Roche, Indianapolis, Ind.) according to themanufacturer's instruction as described (35) with minor modification forsample preparation. In brief, myocytes were rinsed with PBS three timesin order to remove nucleosomes leaked out from necrotic cells and thenincubated with the lysis buffer for 30 min. The supernatant containingmono- and oligonucleosomes from the cytoplasmic fraction of apoptoticcells was used for further analyses. DNA fragmentation of the tissuesamples was determined by DNA laddering assays. Genomic DNA containingsmall molecular weight fragments was isolated and analyzed by agarosegel electrophoresis as described (36).

Immunoblot Analysis

For analyses of Mst1, cells were lysed in a Lysis Buffer A, containing25 mmol/L NaCl, 25 mmol/L Tris (pH 7.4), 1 mmol/L Na₃VO₄, 10 mmol/L NaF,10 mmol sodium pyrophosphate, 0.5 mmol/L EGTA, 0.5 mmol/L AEBSF, 0.5μg/mL leupeptin, 0.5 μg/mL aprotinin. Samples containing the equalamount of protein were subjected to SDS-PAGE. Proteins were transferredonto polyvinylidene fluoride microporous membranes (Bio Rad, Hercules,Calif.) and probed with primary antibodies. We used anti-Mst1 monoclonalantibody (Transduction Laboratory, Lexington, Ky.) for detection of thecarboxyl terminus of Mst1. Affinity purified rabbit polyclonal antibodywas raised against amino-terminal ETVQLRNPPRRQLKC (pAb-15) (BioSourceInternational, Camarillo, Calif.) for detection of the amino-terminus ofMst1. Blots were then probed by horseradish peroxidase-conjugatedanti-mouse IgG or anti-rabbit IgG (Cell Signaling Technolog Inc.,Beverly, Mass.). Antigen-antibody complexes were visualized by theenhanced chemiluminescence system (ECL, Amersham/Pharmacia, Piscataway,N.J.). Polyclonal antibodies raised against cleaved caspase-3 (CellSignaling Technology Inc.) were used to determine activation ofcaspase-3, as described (35).

In Gel Kinase Assay

In gel myelin basic protein (MBP) kinase assays were performed asdescribed previously (38). Either 1000g of total cell lysates, hearthomogenates, or immunoprecipitates of Mst1 were used.Immunoprecipitation of Mst1 was performed by incubating cell extracts(300 □g) with pAb-15 at 4° C. for 3 h followed by 40 □l slurry (50% v/v)of protein A at 4° C. for 1 h. The samples were washed with the lysisbuffer (700 □L) for three times. The immunoprecipitates were boiled withthe Laemmli's SDS-PAGE sample buffer before loading to SDS-PAGE gel.

Transgenic Mice

Mst1 and dominant negative Mst1 (DN-Mst1) transgenic mice (hereafterdesignated as Tg-Mst1 and Tg-DN-Mst1, respectively) were generated(C57BL/6 background) using a cDNA of human myc-Mst1 and myc-Mst1(K59R)driven by the □-myosin heavy chain promoter (courtesy, Dr. J Robbins,University of Cincinnati) to achieve cardiac specific expression.

Echocardiography

Mice were anesthetized with an intraperitoneal injection of ketamine(0.065 mg/g), acepromazine (0.02 mg/g) and xylazine (0.013 mg/g).Echocardiography was performed using ultrasonography (Apogee CX-200;Interspec Inc., Ambler, Pa.) as described previously (39). A dynamicallyfocused 9-MHz annular array transducer was applied from below, using awarmed saline bag as a standoff. M-mode measurements of LV internaldiameter were made from more than 3 beats and averaged. Measurements ofend-diastolic diameter (LVEDD) were taken at the time of the apparentmaximal LV diastolic dimension, while measurements of the LVend-systolic diameter (LVESD) were taken at the time of the mostanterior systolic excursion of the posterior wall. LV ejection fraction(LVEF) was calculated by the cubed method as follows:LVEF=[(LVEDD)³−(LVESD)³]/(LVEDD)³.

Histological Analyses

The heart specimens were fixed with formalin, embedded in paraffin, andsectioned at 6 μm thickness. Interstitial fobrosis was evaluated bypicric acid sirius red staining as described (40). Myocytecross-sectional area was measured from images captured fromsilver-stained 1-μm-thick methacrylate sections as described (36,39,40).Suitable cross-sections were defined as having nearly circular capillaryprofiles and circular-to-oval myocyte sections. No correction foroblique sectioning was made. The outline of 100-200 myocytes was tracedin each section. The MetaMorph image system software was used todetermine myocyte cross-sectional area (36,39,40). The number of myocyte(N) in the sampled area (A) was measured according to the criteriadescribed by Gundersen (48) using Image-Pro Plus software and themyocyte density was calculated as N/A. Using the histologicallydetermined thickness of the LV free wall and the myocyte density perunit area of myocardium, the average number of myocytes across theventricular wall was computed as described (49).

Evaluation of Apoptosis in Tissue Sections

DNA fragmentation was detected in situ by using terminaldeoxyribonucleotide transferase (TdT)-mediated dUTP nick end-labeling(TUNEL) as described (36,39). Briefly, deparaffinized sections wereincubated with proteinase K and DNA fragments were labeled withfluorescein-conjugated dUTP using TdT (Roche). Nuclear density wasdetermined by manual counting of DAPI stained nuclei in 6 fields of eachanimal using the 40× objective, and the number of TUNEL positive nucleicounted by examining the entire section using the same power objective.Limiting the counting of total nuclei and the TUNEL-positive nuclei toareas with true cross section of myocytes made it possible toselectively count only those nuclei that clearly were within myocytes(36). For some samples, triple staining with DAPI, TUNEL, and MF20, andsubsequent analyses using confocal microscopy were performed.

cDNA Expression Array Analyses

DNA microarray analyses were performed by using Atlas cDNA expressionarrays (Clontech Mouse 1.2 Array-II) according to the manufacturer'sinstruction. For each analysis, mRNA was prepared from two hearts ofeither Tg-Mst1 or non-transgenic mice. Analyses of the expressionpattern were conducted by using AtlasNavigator™ 2.0 (Clontech). Analyseswere repeated three times, using three different sets of mRNA samples.

Isolation of Adult Mouse Cardiac Myocytes

Cardiac myocytes were isolated as described previously (41).Longitudinal length of ventricular cardiac myocytes was determined asdescribed (41). Whole cell currents were recorded using patch-clamptechniques (50). Cell capacitance was measured using voltage ramps of0.8V/s from a holding potential of −50 mV.

Measurement of Contractile and Relaxation Function

Cardiac myocytes were isolated as described previously (41). In brief,the heart was rapidly excised and submerged in Ca²⁺-free Tyrode'ssolution containing: 140 mmol/L NaCl, 5.4 mmol/L KCl, 1 mmol/L MgCl₂,0.33 mmol/L Na₂H₂PO₄, 10 mmol/L glucose, 5 mmol/L HEPES (pH7.4). Theheart was digested using 75 U/mL each of collagenase 1 and 2(Worthington) at 32±2° C. All solutions were continuously bubbled with95% O₂ and 5% CO₂ during digestion.

Myocyte contraction was induced once per second (1 Hz) and its functionwas measured using a video motion edge detector (VED 103, CresentElectronics) as described previously (41). The contractile property wasdetermined by % contraction, while the relaxation property by TL70% (thetime for 70% relaxation).

I/R Surgery In Vivo

Mice were anesthetized by intraperitoneal injection of pentobarbitalsodium (60 mg/kg). A rodent ventilator (model 683, Harvard Apparatus,Holliston, Mass.) was used with 65% oxygen during the surgicalprocedure. Ventilation was provided by passing a 20-gauge catheter intothe trachea. The animals were kept warm by using heat lamps and heatingpads. Rectal temperature was monitored and maintained between 36.8 and37.2° C. The chest was opened by a horizontal incision through themuscle between the ribs (3rd intercostal space). Ischemia was achievedby ligating the anterior descending branch of the left coronary artery(LAD) by using a 8-0 nylon suture, with a silicon tubing (1 mm OD)placed on top of the LAD, 2 mm below the border between left atrium andleft ventricle. Regional ischemia was confirmed by ECG change (STelevation). After occlusion for 20 min, the silicon tubing was removedto achieve reperfusion. The chest wall was closed by a 8-0 silk. Theanimal was removed from the ventilator and kept warm in the cagemaintained at 37° C. overnight. Hearts were harvested after 24 hours ofreperfusion.

Assessment of Area at Risk and Infarct Size

After I/R, the animals were re-anesthetized, intubated and chest wasopened. After arresting the heart at the diastolic phase by KClinjection, the ascending aorta was canulated and perfused with saline towash out blood. The LAD was occluded with the same suture, which hadbeen left at the site of the ligation. To demarcate the ischemic area atrisk (AAR), Alcian blue dye (1%) was perfused into the aorta andcoronary arteries. Hearts were excised and LVs were sliced into 1-mmcross sections. The heart sections were then incubated with a 1%triphenyltetrazolium chloride (TTC) solution at 37° C. for 10 minutes.Viable myocardium stained red, and the infarct appeared pale. Theinfarct area (pale), the AAR (not blue), and the total LV area from bothsides of each section were measured by using Adobe Photoshop software,and the values obtained were averaged. The weight of each section wasmeasured by using a balance (Mettler, Columbus, Ohio). The percent areaof infarction and AAR of each section were multiplied by the weight ofthe section and then totaled from all sections. AAR/LV and infarctarea/AAR were calculated and expressed as a percentage. There was nosignificant difference in AAR/LV between Tg-DN-Mst1 and non-transgeniclittermate control.

Statistics

All data are reported as mean ±SEM. Statistical analyses between groupswere done by one-way (ANOVA), and when F values were significant at a95% confidence limit, differences among group means were evaluated usingFisher's project least significant difference post-test procedure forgroup data with a p<0.05 considered significant.

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This invention may be embodied in other forms or carried out in otherways without departing from the spirit or essential characteristicsthereof. The present disclosure is therefore to be considered as in allaspects illustrate and not restrictive, the scope of the invention beingindicated by the appended Claims, and all changes which come within themeaning and range of equivalency are intended to be embraced therein.

Various references are cited throughout this Specification, each ofwhich is incorporated herein by reference in its entirety.

1. A method of treating cardiac disease in a mammal comprising administering to said mammal an effective amount of a compound or agent that blocks or otherwise inhibits Mst1 or the Mst1 pathway.
 2. The method of claim 1 wherein said compound or agent is an Mst1 inhibitor.
 3. The method of claim 1 wherein the Mst1 inhibitor is selected from the group of a dominant negative mutant of Mst1, an inhibitor of caspase, an inhibitor of apoptosis, antisense oligonucleotide complementary to Mst1, siRNA to Mst1 and a ribozyme directed against Mst1.
 4. The method of claim 3, wherein said Mst1 inhibitor is selected from the group of inhibitors against Caspase 3, Chelerythrine and Calyculin A.
 5. The method of claim 1 wherein said compound or agent inhibits a protein that phosphorylates Mst1, cleaves Mst1 to generate active Mst1, or activates an Mst1 phosphatase.
 6. The method of claim 1 wherein said cardiac disease is selected from the group of congestive heart failure, cardiomyopathy, including ischemic and nonischemic cardiomyopathy, coronary artery disease, arrhythmias, fibrosis of the heart, valve defects, atherosclerosis, and instances where facilitation of enhanced heart function or maintenance of cardiac myocytes is desired.
 7. The method of claim 1 wherein said mammal is a human.
 8. A method of modulating cardiac myocyte apoptosis in a mammal comprising administering to said mammal an effective amount of a compound or agent that blocks or otherwise inhibits Mst1 or the Mst1 pathway.
 9. The method of claim 8 wherein the Mst1 inhibitor is selected from the group of a dominant negative mutant of Mst1, an inhibitor of caspase, an inhibitor of apoptosis, antisense oligonucleotide complementary to Mst1, siRNA to Mst1 and a ribozyme directed against Mst1.
 10. A method for reducing cardiomyopathy in a mammal comprising administering to said mammal an effective amount of an Mst1 inhibitor.
 11. A method for treating cardiac disease in a mammal comprising administering to said mammal an effective amount of a Mst1 inhibitor in combination with one or more other compound for treatment of cardiac disease or of atherosclerosis.
 12. The method of claim 11 wherein said one or more other compound selected from the group of a beta-blocker, nitrate, calcium channel antagonists, angiotensin-converting enzyme (ACE) inhibitors, an anti-platelet drug, diuretics, digoxin and antilipemic agents, agents which alter cholesterol or lipid metabolism.
 13. A method for reducing the risk of cardiomyopathy or cardiac dysfunction in a mammal wherein said mammal has suffered a myocardial infarct or other coronary event wherein blood flow to the heart is reduced comprising administering to said mammal an effective amount of an Mst1 inhibitor or Mst1 pathway inhibitor.
 14. The method of claim 13 wherein the Mst1 inhibitor is selected from the group of a dominant negative mutant of Mst1, an inhibitor of caspase, an inhibitor of apoptosis, antisense oligonucleotide complementary to Mst1, siRNA to Mst1 and a ribozyme directed against Mst1.
 15. A method of cardioprotection, wherein an inhibitor of Mst1 is administered in conjunction with or following therapy with a compound or drug which is cardiotoxic.
 16. The method of claim 15 wherein said compound is a chemotherapeutic agent, particularly an anti-cancer or anti-tumor agent.
 17. The method of claim 16 wherein said chemotherapeutic agent is doxorubicin.
 18. A method of screening for compounds which modulate cardiac myocyte apoptosis comprising selecting compounds which inhibit Mst1 or the Mst1 pathway and performing assays with said compounds to determine the amount or extent of cardiac myocyte apoptosis.
 19. The method of claim 18 wherein selecting compounds that modulate Mst1 or the Mst1 pathway comprises incubating Mst1 with a candidate compound, and conducting phosphorylation assays wherein a compound's ability to block phosphorylation of Mst1 or by Mst1 or enhance dephosphorylation of Mst1 is determined.
 20. The method of claim 18 wherein selecting compounds that modulate Mst1 or the Mst1 pathway comprises incubating Mst1 with a candidate compound, and conducting assays wherein a compound's ability to block the activity of a molecule downstream of Mst1 or which is modulated or activated by Mst1 or upon Mst1 phosphorylation or cleavage is determined.
 21. The method of claim 20 wherein the molecule downstream of Mst1 is selected from the group of caspase 3, caspase 5, MBP, MAPK and JNK.
 22. A composition for modulating cardiac myocyte apoptosis comprising an Mst1 inhibitor.
 23. The composition of claim 22 wherein said Mst1 inhibitor inhibits a protein that phosphorylates Mst1, activates an Mst1 phosphatase, inhibits a protein that cleaves and/or activates Mst1, is an antagonist of Mst1, or is a dominant negative form Mst1.
 24. A pharmaceutical composition for treatment or amelioration of cardiac disease in a mammal comprising a therapeutically effective amount of one or more Mst1 inhibitor and a pharmaceutically acceptable carrier.
 25. A pharmaceutical composition for treatment or amelioration of cardiac disease in a mammal comprising a therapeutically effective amount of a combination of one or more Mst1 inhibitor and one or more other compounds for the treatment of cardiac disease or atherosclerosis and a pharmaceutically acceptable carrier.
 26. The pharmaceutical composition of claim 25 wherein the one or more other compounds for the treatment of cardiac disease or atherosclerosis are selected from the group of a beta-blocker, nitrate, calcium channel antagonists, angiotensin-converting enzyme (ACE) inhibitors, an anti-platelet drug, diuretics, digoxin and antilipemic agents, agents which alter cholesterol or lipid metabolism.
 27. The pharmaceutical composition of claim 25 or 26 wherein the cardiac disease is selected from the group of congestive heart failure, cardiomyopathy, including ischemic and nonischemic cardiomyopathy, coronary artery disease, arrhythmias, fibrosis of the heart, valve defects, atherosclerosis, and instances where facilitation of enhanced heart function or maintenance of cardiac myocytes is desired.
 28. An assay system for screening of potential compounds or agents effective to modulate Mst1 activity of target mammalian cells by interrupting or potentiating the Mst1 or Mst1 pathway wherein the test compound or agent is administered to a cellular sample to determine its effect upon the kinase activity, cleavage status or phosphorylation status of Mst1, by comparison with a control.
 29. An assay system for screening compounds or agents for the ability to modulate the activity of Mst1, comprising: A. culturing an observable cellular test colony inoculated with a compound or agent; B. harvesting a supernatant from said cellular test colony; and C. examining said supernatant for the activity of said Mst1 wherein an increase or a decrease in the activity of said Mst1 indicates the ability of a drug to modulate the activity of said Mst1.
 30. A method for treating or ameliorating cardiac disease in a mammal comprising administering to said mammal a nucleic acid or vector capable of encoding a dominant negative form of Mst1 such that said dominant negative form of Mst1 is expressed in the heart of said mammal.
 31. The method of claim 30 wherein the dominant negative form is expressed in cardiac myocytes and wherein the dominant negative form of Mst1 antagonizes the endogenous form of Mst1 such that Mst1 is inhibited.
 32. An animal model of cardiac disease, including cardiac myopathy, comprising a transgenic animal wherein Mst1 expression or activity is enhanced.
 33. The animal model of claim 32 wherein the transgenic animal is selected from the group of rats, mice, pigs, chicken, cows, monkeys, rabbits, sheep and dogs. 